EP4619764A1 - Igfbp2 as biomarker for thoracic aortic aneurysm and dissections - Google Patents

Abstract

The present invention provides methods for detecting thoracic aortic aneurysms and thoracic aortic dissections. In particular, the present invention provides an in vitro method for detecting a thoracic aortic aneurysm in a subject which relies on determining the level of Insulin-like Growth Factor Binding Protein-2 (IGFBP2) in a biological sample of the patient. The invention further provides a kit of parts for conducting the methods subject of the invention, and methods to monitor clinical progression of thoracic aortic aneurysm and assessing the efficacy of a therapeutic treatment directed against thoracic aortic aneurysm.

Description

IGFBP2 AS BIOMARKER FOR THORACIC AORTIC ANEURYSM

AND DISSECTIONS

FIELD OF THE INVENTION

The present invention relates broadly to the field of molecular diagnostics. Particularly, the invention relates to Insulin-like Growth Factor Binding Protein-2 (IGFBP2) as a biomarker for thoracic aortic aneurysm and dissection. The invention concerns methods for detecting thoracic aortic aneurysm and dissection based on a detected IGFBP2 expression level. Further described are methods for monitoring clinical progression of said disease and for assessing the efficacy of a therapeutic treatment of said disease.

BACKGROUND OF THE INVENTION

The aorta is the main and largest blood vessel in the body and distributes oxygenated blood to all parts of the body. The aorta originates from the left ventricle of the heart and extends down to the abdomen where it forms the common iliac arteries. Since the aorta supplies all of the systemic circulation, it is evident that a proper functioning and structural integrity of the aorta is crucial for any person. Anatomically, the aorta is divided into a thoracic and abdominal component, respectively above and below the diaphragm. The thoracic aorta includes the aortic root and the ascending aorta, the aortic arch and the descending segments.

Aortic aneurysms, which involve the formation of a local bulge in the aortic wall (i.e. ballooning), weaken the strength of said wall. The force of the blood moving through the vessel can lead to an aneurysm. Over time, without treatment, the aneurysm can grow and either split (dissection) or rupture, often leading to death. Aortic aneurysm is the second most common disease affecting the aorta after atherosclerosis and the fifteenth leading cause of death in individuals over 55 years of age (Erbel et al., Eur Heart J, 2014; and Centers for Disease Control and Prevention: Leading causes of death and injury, CDC, 2020). Despite improvements in surgical repair, the morbidity and mortality of aneurysms remain high, especially for thoracic aortic aneurysms.

The estimated incidence of thoracic aortic aneurysms (TAA) is 5 to 10 per 100,000 person-years (Kuzmik et al., J Vase Surg, 2012). This number has been gradually increasing over time, which may to a certain extent be attributed to the ageing of the general population and increased use of advanced imaging techniques that facilitate detection. Given that a large majority of patients remain asymptomatic until a catastrophic event happens, the incidence of thoracic aortic aneurysms is likely to be vastly underestimated. Thus, thoracic aortic aneurysm and dissection (TAAD) represents a major cause of mortality and morbidity worldwide, and especially in the Western world. At present, there is not a single biomarker for thoracic aortic aneurysm and/or dissection available in clinical practice. Although certain molecules such as cGMP and nitrated proteins were found to be elevated in patients diagnosed with the Marfan Syndrome, their role as general biomarker for TAAD has not been shown (WO 2022/219196). Diagnosis of aortic aneurysm/dissection fully relies on imaging techniques (e.g. positron emission tomography (PET), echocardiography, computerised tomography (CT)-scanning, or Magnetic Resonance Imaging (MRI)). Whilst effective, these methods often require a separate medical appointment, specialized medical personnel, and adequate infrastructural capacity to ensure an acceptable patient throughput. In view hereof, there is an unmet high need for a clinically useful biomarker for thoracic aortic aneurysmal disease to be able to prevent the fatal consequences of an aortic dissection or rupture by allowing an early intervention (Bossone and Eagle, Nat Rev Cardiol, 2021).

SUMMARY OF THE INVENTION

The present inventors have identified a biomarker that allows for accurately predicting TAA and/or TAD development in a subject, irrespective of any underlying genetic cause by initially performing a hypothesis-free bulk mRNA-sequencing approach of aortic wall tissue in three genetically modified mouse models (F ^C1O41G/+, Ipo8~'~, Smad3~'~) presenting with thoracic aortic aneurysm at 16 weeks of age. mRNA expression profiling of the aortic wall of these mice and their respective wild-type littermates revealed a significant consistent upregulation of IGFBP2 (insulin-like growth factor binding protein 2) in all three models. This finding is of particular importance since IGFBP2 expression is known to be the highest in the aorta (GTEx bulk tissue gene expression dataset (https://www.gtexportal.org)). This expression profile is also specific for the vasculature with significantly lower or no detectable expression in other tissues.

In addition to the finding that IGFBP2 in biological samples (e.g. serum samples) could serve as a specific biomarker for aortic aneurysm and/or aortic dissection development, IGFBP2 serum concentrations could be correlated to thoracic aortic aneurysm progression and severity in Marfan (F/w7^cl041G/+) and Loeys-Dietz syndrome (Ipo8~'~, SmadS'¹') mouse models. Finally IGFBP2 levels can also detect asymptomatic TAA, monitor disease progression and predict aortic rupture and/or dissection.

Accordingly, a first aspect of the invention provides an in vitro method for the detection of thoracic aortic aneurysm (TAA) and/or thoracic aortic dissection (TAD) in a subject. More particularly, the method comprises determining the level of IGFBP2 expression in a biological sample of said subject.

In particular embodiments, the biological sample is a serum sample of the subject. Preferred subjects in the context of the invention are human subjects. In preferred embodiments, the IGFBP2 is human IGFBP2 optionally characterized by SEQ ID NO: 1 (see below). In particular embodiments, the method comprises comparing said level of IGFBP2 expression in a biological sample and determining whether said IGFBP2 level is altered when compared to a control sample. In particular embodiments, the level of IGFBP2 expression is normalized based on a detected housekeeper genes in the biological sample and/or the control sample.

In particular embodiments, the method comprises determining whether the IGFBP2 level is increased when compared to a control sample.

In particular embodiments, the thoracic aortic aneurysm involves the aortic root aorta, the ascending aorta, the aortic arch, the descending aorta, the thoracoabdominal aorta, or a combination thereof.

In particular embodiments, the control sample is a sample obtained from an individual having normal aortic diameters or no aortic dissection.

In particular embodiments, the expression level of IGFBP2 is indicative for the severity and/or speed of development of the thoracic aortic aneurysm and/or thoracic aortic dissection. In particular embodiments, a relatively higher expression level of IGFBP2 is indicative for a relatively higher severity and/or speed of development of the thoracic aortic aneurysm or thoracic aortic dissection.

In particular embodiments, the method is a method of detecting asymptomatic thoracic aortic aneurysm, asymptomatic thoracic aortic dissection, and/or symptomatic thoracic aortic dissection in a subject. In further embodiments, the subject is an asymptomatic subject to which the method subject of the invention is applied in a screening context.

In particular embodiments, the method is a method of monitoring disease progression in a subject diagnosed with thoracic aortic aneurysm and/or thoracic aortic dissection.

In particular embodiments, the method is a method of predicting aortic dissection in said subject.

In particular embodiments, the subject is a subject characterized by the presence of one or more risk factors. In further embodiments the one or more risk factors are selected from the group consisting of: aging (i.e. the subject is an elderly subject), atherosclerosis, blunt chest trauma, elevated blood pressure (i.e. hypertension), bicuspid aortic valve, coarctation (narrowing) of the aorta, connective tissue disorders (such as Marfan syndrome and Ehlers-Danlos syndrome) and rare genetic disorders, heart surgery, pregnancy, arteritis, syphilis, substance abuse (e.g. cocaine or methamphetamine abuse), periodical weight lifting (e.g. bodybuilding), smoking, diabetes, medication use, and hypercholesterolemia. In particular embodiments, the method comprises an additional step of applying a normalization coefficient on the IGFBP2 expression level based on the occurrence of one or more of the above-mentioned risk factors. In particular embodiments, the method further comprises determining whether said subject has a genetic predisposition to developing thoracic aortic aneurysm and/or thoracic aortic dissection.

In particular embodiments, the subject is a subject with bicuspid aortic valves (BAV), idiopathic degenerative disease, or a tricuspid aortic valve (TAV).

In particular embodiments, the method further comprises detecting one or more of additional biomarkers in the biological sample of said subjects.

In particular embodiments, the method further comprises treating said subject with one or more medicaments which can slow down said thoracic aortic aneurysm development and/or thoracic aortic dissection. Optionally, the method additionally comprises a step of assessing the efficacy of said one or more medicaments by means of IGFBP2 expression level monitoring (i.e. measuring IFGBP2 expression levels on at least two distinct time points). In further embodiments, the one or more medicaments are selected from the group consisting of: beta blockers, angiotensin II receptor blockers, and statins.

Hence, in particular aspects the method allows for assessing the efficacy of one or more medicaments in a context of thoracic aortic aneurysm or dissection by assessing IGFBP2 expression levels. Related hereto, assessment of IGFBP2 may also provide a skilled practitioner with an indication about the suitability of one or medicaments for treating thoracic aortic aneurysm or dissection.

In particular embodiments, the method comprises determining the level of Igfbp2 protein and/or the level of IGFBP2 mRNA present in said biological sample.

In particular embodiments, the level of IGFBP2 is detected by a biochemical method, immunoassay method, mass spectrometry analysis method, chromatography method, or any combination thereof. In particular embodiments, the IGFBP2 expression level is measured on the transcript (mRNA) level by any suitable technology, including without limitation quantitative polymerase chain reaction, microarray assay, RNA-sequencing, or any combination thereof. The IGFBP2 expression level may equally be measured on the protein level by measurement of the expression levels of the translated proteins by any suitable technology, including but not limited to technologies such as mass spectrometry, (Western) blotting, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, or any combination thereof. Optionally, the IGFBP2 expression level may be measured both on the transcript level and on the protein level.

In a further aspect, the invention is directed to a kit of parts for detecting IGFBP2 and conducting the diagnostic method described herein.

In yet a further aspect, measuring the expression level of IGFBP2 is envisaged for detecting a developing thoracic aortic aneurysm and/or thoracic aortic dissection. In a further aspect the present invention provides a kit of parts for predicting thoracic aortic aneurysm development comprising means for measuring the expression level of IGFBP2 in a biological sample such as a serum sample. In particular embodiments, the kit of parts may comprise one or more suitable control samples such as a negative control and/or a positive control.

The above and further aspects and preferred embodiments of the invention are described in the following sections and in the appended claims. The subject matter of the appended claims is hereby specifically incorporated in this specification.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. RT-qPCR results for IGFBP2 expression. Different genetic mouse backgrounds are tested for IGFBP2 expression.

Figure 2. ROC curve of the preliminary age-corrected IGFBP2 ELISA results.

Figure 3. IGFBP2 concentration in control (healthy) subjects versus IGFBP2 concentration in dissection subjects as measured by ELISA.

DETAILED DESCRIPTION

As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

The terms “comprising”, “comprises” and “comprised of’ as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms also encompass “consisting of’ and “consisting essentially of’, which enjoy well-established meanings in patent terminology.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints. This applies to numerical ranges irrespective of whether they are introduced by the expression “from. . . to. . . ” or the expression “between. . . and. . . ” or another expression.

The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of +/-10% or less, preferably +/-5% or less, more preferably +/- 1% or less, and still more preferably +/-0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed. Whereas the terms “one or more” or “at least one”, such as one or more members or at least one member of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g. any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members. In another example, “one or more” or “at least one” may refer to 1, 2, 3, 4, 5, 6, 7 or more.

The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge in any country as of the priority date of any of the claims.

Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings or sections of such documents herein specifically referred to are incorporated by reference.

Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the invention. When specific terms are defined in connection with a particular aspect of the invention or a particular embodiment of the invention, such connotation or meaning is meant to apply throughout this specification, i.e. also in the context of other aspects or embodiments of the invention, unless otherwise defined. For example, embodiments directed to products are also applicable to corresponding features of methods and uses.

In the following passages, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

Reference throughout this specification to “one embodiment”, “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, alternative combinations of claimed embodiments are encompassed, as would be understood by those in the art.

Unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person. Reference is for example again made to standard handbooks as well as to the general background art referred to herein and to the further references cited therein.

Any genes of interest disclosed in the context of the findings made by the inventors are in addition to their full name indicated by their commonly accepted GeneCards Symbol (https://www.genecards.org/). A skilled person appreciates that described herein may equally be annotated by alternative identifiers such as but not limited to their HGNC (https://www.genenames.org/), NCBI Entrez Gene (https://www.ncbi.nlm.nih.gov/gene/), Ensembl (http://www.ensembl.org/), or UniProtKB identifier (https://www.uniprot.org). A skilled person readily appreciates that any sequences represented in sequence databases may be of precursors of markers, peptides, polypeptides, proteins, or nucleic acids and may include parts which are processed away from mature molecules.

The thoracic aorta may be described as a tube composed of three distinct layers: an inner layer (tunica intima), a middle layer (tunica media), and an outer layer (tunica adventitia). The inner layer comprises predominantly endothelial cells. The middle layer is predominantly composed of smooth muscle cells and elastin interspersed by collagen fibres and proteoglycan-rich extracellular matrix. The outer layer is rich in collagen extracellular matrix and fibroblasts. The middle layer of the aorta confers elasticity and strength to the aortic wall, and is composed of >50 alternating layers of smooth muscle cells and elastic lamellae in humans. The smooth muscle cell-elastin-contractile unit is the structural unit that connects the elastin lamellae to the smooth muscle cells. Microfibrillar extensions from the elastic lamellae are connected in a diagonal manner to the surface of the smooth muscle cells through dense plaques and provide a connection of the smooth muscle cells to the elastic fibres, ultimately allowing the propagation of mechanical forces between elastin and smooth muscle cells via integrin receptors.

This elastin-contractile unit is uniquely designed to coordinate smooth muscle cell contractions and elastic tensions in response to mechanical stress exerted on the vessel wall origination from pulsatile blood flow (Pinard et al., Circ Res, 2019).

The inventors have found that IGFBP2 is significantly upregulated in aortic wall tissue of multiple genetically modified mouse models (F ^C1O41G/+, IpoB'~, Smad3~'~) that each are characterized by the occurrence of thoracic aortic aneurysm at 16 weeks of age. mRNA expression profiling of the aortic wall of these mice and their respective wild-type littermates revealed a significant consistent upregulation of IGFBP2 in all three models. Importantly, according to the GTEx bulk tissue gene expression dataset (https://www.gtexportal.org), IGFBP2 expression is highest in the aorta. The expression profile is also specific for the vasculature with significantly lower or no detectable expression in other tissues. Earlier investigations in animal and cell models have revealed a role for IGFBP2 in vascular smooth muscle cell proliferation, vascular smooth muscle cell migration, and angiogenesis, as well as a functional relationship with several molecules that have been linked to thoracic aortic aneurysm development before (such as but not limited to Sirtl, Adamtsl and pErkl/2) (Slater et al., Heart, 2019). Targeted IGFBP2 knock-down in zebrafish embryos resulted in angiogenic defects and cardiovascular development disruption (Wood et al., Mol Endocrinol, 2005). In vascular smooth muscle cells isolated from pig aorta, it was demonstrated that IGFBP2-potentiated IGF1- induced vascular smooth muscle cell migration and proliferation through interaction with receptor protein phosphatase beta (Hsieh et al., J Biol Chem, 2003). Transgenic overexpression o Igfbp2 in mice demonstrated attenuated aortic contractility independent of NO. Contractile vascular smooth muscle cell markers were downregulated in these mice, suggesting a switch from contractile to synthetic phenotype. A more recent scRNA sequencing study of aortic wall tissue of patients with monogenic aortopathies (ACTA J. FBN1, TGFBR1, TGFB2, SMAD3) suggested that IGFBP2 is a marker of early smooth muscle cell modulation in human aortopathy. Finally, IGFBPs are known to modulate IGF- driven endothelial cells migration and vasodilatation through endothelial cell secretion of nitric oxide. As such the inventors hypothesized that IGFBP2 in serum could serve as a specific biomarker for aortic aneurysm development, irrespective of the underlying (genetic) cause. Moreover, quantitation of the IGFBP2 levels in for example serum samples correlate to a certain extent with disease severity. The present findings thus provide evidence that for example IGFBP2 enzyme-linked immunosorbent assay (ELISA) measurements in serum of subjects is a powerful method to screen and follow-up on thoracic aortic aneurysm development, as well as providing a suitable means for monitoring therapy strategies.

In the art, IGFBP2 activation has been reported as a marker of smooth muscle cells (SMCs) in transition from one subtype to another (e.g. Pedroza et al., Circulation, 2021). It was hypothesized that IGFBP2 activation may promote the contractile phenotype, whereby IGFBP2 activation was identified as a marker for only a subpopulation of SMCs. They suggest that IGFBP2 expression is indicative of susceptibility to aortopathy but do not link this marker to a specific disease or disease severity. Moreover, in the pathogenesis of TAA, SMC contractility is widely believed to be impaired.

The role of IGFBP2 in heart failure has also been evaluated (e.g. Barutaut et al., International Journal of Cardiology, 2020). However, as further described in detail below there is a clear distinction to be made between heart failure and TAA/TAD. A biomarker for the former (collection of) condition(s) cannot be considered of relevance for the latter condition(s). Accordingly, the invention provides in an in vitro method for the detection of thoracic aortic aneurysm (TAA) and/or thoracic aortic dissection (TAD) in a subject, said method comprising determining the expression level of an insulin-like growth factor binding protein-2 (IGFBP2) in said subject. Highly preferred embodiments of the invention are those wherein the method comprises determination of the expression level of insulin-like growth factor binding protein 2 (IGFBP2) in a biological sample of the subject.

“Insulin-like growth factor binding protein 2”, abbreviated throughout the present disclosure and in the art as “Igfbp2” is a protein that in humans is encoded by the IGFBP2 gene. While generally and in accordance with conventional molecular nomenclature “IGFBP2” refers to the gene and “Igfbp2” refers to the protein, “IGFBP2” as used herein is intended to encompass both the gene and any gene product (i.e. RNA transcript and protein) since the method subject of the invention is applicable on both the transcript and the protein level. Hence, “IGFBP2” and “Igfbp2” can be used interchangeably herein unless explicit reference is made to the gene, transcript, and/or protein form. Alternative names for IGFBP2 used throughout the art include without limitation “IGFBP-2”, “IBP-2”, “BP2” and “IBP2”. Igfbp2 protein is part of the insulin-like growth factor binding proteins (IGFBPs), a family of proteins binding to Insulin-like growth factors (IGFs), generally including IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, and IGFBP6. Igfbp2 protein can be secreted into the bloodstream, where it is capable of binding IGF-I and IGF-II with high affinity, or can remain intracellular where it is capable of interacting with many different ligands. Both the IGFBP2 genomic sequence and protein sequence have been described and are therefore known to a skilled person (reference is made in this regard to UniProt identifier Pl 8065; HGNC identifier HGNC:5471, Esembl identifier ENST00000233809.9, and GenelD identifier 3485). High expression levels of this protein promote the growth of several types of tumours and may be predictive of the chances of recovery of the patient. Several transcript variants, one encoding a secreted isoform and the others encoding non-secreted isoforms, have been identified (i.e. UniProt identifiers C9JW52, C9JMY1, and H7C1H0). Unless explicitly indicated otherwise, any reference to Igfbp2 encompasses both the canonical product and any isoforms. Any references to certain proteins or genes throughout the present disclosure indicate human proteins or human genes unless explicitly stated otherwise. For convenience, the canonical sequence of IGFBP2 (UniProt ID Pl 8065) is reproduced below as SEQ ID NO: 1:

MLPRVGCPALPLPPPPLLPLLLLLLGASGGGGGARAEVLFRCPPCTPERLAACGPPPVAPPAAVAAVA GGARMPCAELVREPGCGCCSVCARLEGEACGVYTPRCGQGLRCYPHPGSELPLQALVMGEGTCEKRRD AEYGASPEQVADNGDDHSEGGLVENHVDSTMNMLGGGGSAGRKPLKSGMKELAVFREKVTEQHRQMGK GGKHHLGLEEPKKLRPPPARTPCQQELDQVLERISTMRLPDERGPLEHLYSLHIPNCDKHGLYNLKQC KMSLNGQRGECWCVNPNTGKLIQGAPTIRGDPECHLFYNEQQEARGVHTQRMQ (SEQ ID NO: 1).

A first isoform of IGFBP2 (UniProt ID C9JW52) is characterised by the sequence SEQ ID NO: 2: MNMLGGGGSAGRKPLKSGMKELAVFREKVTEQHRQMGKGGKHHLGLEEPKKLRPPPARTPCQQELDQV

LERISTMRLP (SEQ ID NO: 2).

A second isoform of IGFBP2 (UniProt ID C9JMY1) is characterised by the sequence SEQ ID NO: 3:

MPCNNGDDHSEGGLVENHVDSTMNMLGGGGSAGRKPLKSGMKELAVFREKVTEQHRQMGKGGKHHLGL EEPKKLRPPPARTPCQQELDQVLERISTMRLPDERGPLEHLYSLHIPNCDKHGLYNLKQCKMSLNGQR GECWCVNPNTGKLIQGAPTIRGDPECHLFYNEQQEARGVHTQRMQ (SEQ ID NO: 3).

A third isoform of IGFBP2 (UniProt ID H7C1H0) is characterised by the sequence SEQ ID NO: 4:

KHHLGLEEPKKLRPPPARQLLAELASGACFVGLLSSLRASPAVCACRLPANRNWTRSWSGSPPCAFRM SGALWSTSTPCTSPTVTSMACTTSNSARCL (SEQ ID NO: 4).

Hence, in certain embodiments, the method comprises detecting, and optionally quantitating the presence of a sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or any combination thereof. In alternative embodiments, the method comprises detecting, and optionally quantitating the presence of a nucleic acid sequence encoding a sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or any combination thereof. In certain embodiments, the method comprises detecting, and optionally quantitating the presence of each of the sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4. In alternative embodiments, the method comprises detecting, and optionally quantitating the presence of nucleic acid sequences encoding each of the sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4.

It is appreciated that a skilled person is capable of assessing sequence identity between sequences. The term "sequence identity" of two sequences as used herein relates to the number of positions with identical nucleotides or amino acids divided by the number of nucleotides or amino acids in the shorter of the sequences, when the two sequences are aligned. Methods and tools to verify sequence identity between different sequences of amino acids or nucleic acids are well known to a person skilled in the art and include (Protein) BLAST, ClustalW2, SIM alignment tool, TranslatorX and T-COFFEE. The percentage of identity between two sequences may show minor differences depending on the algorithm choice and parameters. The term “sequence identity” as used herein refers to the relationship between sequences at the nucleotide (or amino acid) level. The expression “% identical” is determined by comparing optimally aligned sequences, e.g. two or more, over a comparison window wherein the portion of the sequence in the comparison window may comprise insertions and/or deletions as compared to the reference sequence for optimal alignment of the sequences. The reference sequence does not comprise insertions or deletions. A reference window is chosen and the “% identity” is then calculated by determining the number of nucleotides (or amino acids) that are identical between the sequences in the window, dividing the number of identical nucleotides (or amino acids) by the number of nucleotides (or amino acids) in the window and multiplying by 100. Unless indicated otherwise, the sequence identity is calculated over the whole length of the reference sequence. An example procedure to determine the percent identity between a particular amino acid sequence and the amino acid sequence of a query polypeptide will entail aligning the two amino acid sequences using the Blast 2 sequences (B12seq) algorithm, available as a web application or as a standalone executable programme (BLAST version 2.2.31+) at the NCBI web site (www.ncbi.nlm.nih.gov), using suitable algorithm parameters. An example of suitable algorithm parameters include: matrix = Blosum62, cost to open a gap = 11, cost to extend a gap = 1, expectation value = 10.0, word size = 3).

In certain embodiments, the method comprises detecting the expression of an amino acid sequence having at least 65% sequence identity to SEQ ID NO: 1. In further embodiments, the method comprises detecting the expression of an amino acid sequence having at least 75%, preferably at least 85%, more preferably at least 95% sequence identity to SEQ ID NO: 1. In certain embodiments, the method comprises detecting the expression of a nucleic acid sequence encoding an amino acid sequence having at least 65% sequence identity to SEQ ID NO: 1. In further embodiments, the method comprises detecting the expression of a nucleic acid sequence encoding an amino acid sequence having at least 75%, preferably at least 85%, more preferably at least 90%, more preferably at least 92.5%, more preferably at least 95%, more preferably at least 97.5%, more preferably 99%, most preferably at least 99.5% sequence identity to SEQ ID NO: 1.

It is appreciated by the above embodiments that the present invention envisages detection of IGFBP2 variants. The term “variant” of a nucleic acid, protein, polypeptide or peptide refers to nucleic acids, proteins, polypeptides or peptides the sequence (i.e. nucleotide sequence or amino acid sequence, respectively) of which is substantially identical (i.e. largely but not wholly identical) to the sequence of said recited nucleic acid, protein or polypeptide.

It is appreciated that the present disclosure concerns the finding that IGFBP2 expression levels are indicative for thoracic aortic aneurysm (development) and/or thoracic aortic dissection in a subject and that IGFB2 may be considered a biomarker for said conditions. It should be equally appreciated that IGFBP2 expression levels are indicative for an increased risk to develop thoracic aortic aneurysm (development) and/or thoracic aortic dissection.

Reference throughout the present description is made to terms such as “expression”, “expression level”, “quantity”, “amount”, “value”, and “level”, which each indicate a quantification of a gene expression level in a biological sample. Said quantitation may be an absolute or alternatively a relative quantification of a gene expression level in a biological sample. Relative quantification in the present context refers to the expression of a gene expression level relative to another value such as relative to a reference value, or even reference range (e.g. a reference indicating a base-line expression of a marker in a given tissue), or relative to the expression level in a reference biological sample (i.e. indicated interchangeably herein by the term “control sample”). These values or ranges can be obtained from a single biological sample or from a plurality of biological samples (i.e. biological repeats) and may be established by determining the IGFBP2 expression level in biological samples from one subject or from a population of subjects characterised by an existing particular diagnosis, prediction, prognosis and/or monitoring of thoracic aortic aneurysm and/or thoracic aortic dissection. Such a population may comprise without limitation at least 2, preferably at least 10, more preferably at least 100, most preferably at least several hundred subjects or more. In the context of the predictive methods described herein, the status of a subject or population of individuals as to the presence or ongoing development of thoracic aortic aneurysm and/or thoracic aortic dissection may not be known at the time of sampling said subject or population of subjects, but will become known later on, such that the reference value generated on the basis of said subject or population of subjects can then be allocated to the particular prediction of thoracic aortic aneurysm and/or thoracic aortic dissection as observed in said subject or population of subjects.

The value may be obtained by a single measurement of the gene expression level in a biological sample, or by repeated measurement of the gene expression level in a biological sample (i.e. technical repeats).

A suitable means to determine an optimal threshold value for IGFBP2 expression in a population of subjects for clinical use of the method described herein is a receiver-operating characteristic (ROC) curve analysis based on acceptable sensitivity and specificity, or related performance measures which are well-known in the art, such as but not limited to positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (LR+), negative likelihood ratio (LR-), Youden index, or similar.

Optionally, the method may comprise a step of normalising the expression levels of IGFBP2 in the biological sample of the subject to the expression level of one or more housekeeper genes. In further embodiments, the method may comprise a step of normalising both the expression levels of the genes in the biological sample of the subject and the reference expression levels of the genes when the references values are obtained from a control sample to the expression level of one or more housekeeper genes. The expression level of the one or more housekeeper genes are preferably measured in the same biological sample, or same control sample wherein the expression levels of the genes are measured.

“Housekeeping genes” are genes that are characterised by a constant, or essentially constant expression level in a group of tissues and/or a group of subjects (more specifically in a biological sample thereof). The term is well known to a person skilled in molecular biology and a skilled person thus appreciates that reference thereto implies a reference to one or more genes having uniform or near uniform expression levels with low variance between different samples, said samples optionally being subjected to different conditions and/or treatments. In the context of the present disclosure, suitable housekeeping genes are genes that have uniform or near uniform expression levels and are invariable or essentially invariable to changes in temperature. In addition, a skilled person is aware of repositories compiling housekeeping genes such as but not limited to the “Housekeeping and Reference Transcript Atlas” (Hounkpe et al., Nucleic Acids Res, 2021). Cellular localisation and function of genes are not particularly limiting to act as a housekeeping gene for normalisation of gene expression levels, as the suitability is solely determined by their (near) constant expression levels. Hence, suitable housekeeping genes may be identified in any of the following non-limiting gene categories: genes regulating gene expression, genes involved in metabolism, genes encoding structural cellular components, genes encoding surface proteins, kinase genes, signalling genes, etc.

An absolute quantitation of a gene expression level in a biological sample may be expressed by various measurement units such as but not limited to weight, molar amount, concentration (e.g. weight per volume or mol per volume), intensity, or copy number. A relative quantitation of a gene expression level in a biological sample may be expressed by various measurement units such as but not limited to increase, decrease, fold-increase, or fold-decrease relative to a reference gene expression level.

The term “biomarker” in the context of the present disclosure encompasses any physical form of the IGFBP2 biomarker including proteins, polypeptides, peptides, nucleic acids, and any metabolic products thereof. Additionally, the term “biomarker”, often indicated in the art by the term “marker”, is widespread in the art and commonly broadly denotes a biological component or a biological molecule, more particularly an endogenous biological component or molecule, or a detectable portion thereof, whose qualitative and/or quantitative evaluation in a tested subject, such as by means of evaluating a biological sample from the subject, is predictive (e.g. predictive, diagnostic and/or prognostic) or informative with respect to one or more aspects of the tested subjects’ phenotype and/or genotype for example with respect to the status of the subject as to a given disease or condition, in the context of the present invention thoracic aortic aneurysm and/or thoracic aortic dissection. A skilled person understands that native sequences may differ between different species due to genetic divergence between such species. Moreover, native sequences may differ between or within different individuals of the same species due to normal genetic diversity (variation) within a given species. Also, native sequences may differ between or even within different individuals of the same species due to post- transcriptional or post-translational modifications. Any such variants or isoforms of markers, peptides, polypeptides, proteins, or nucleic acids are intended herein. Accordingly, all sequences of markers, peptides, polypeptides, proteins, or nucleic acids found in or derived from nature are considered “native”. The terms encompass the markers, peptides, polypeptides, proteins, or nucleic acids when forming a part of a living organism, organ, tissue or cell, when forming a part of a biological sample, as well as when at least partly isolated from such sources. The terms also encompass markers, peptides, polypeptides, proteins, or nucleic acids when produced by recombinant or synthetic means. Optionally and as described further herein, the IGFBP2 biomarker may be combined with additional biomarkers and in these embodiments expressions such as but not limited to “a plurality of biomarkers” is indicative for at least two biomarkers. Said expression may be used interchangeably with related terms and expressions such as but not limited to “a biomarker signature”, “a set of biomarkers”, or “a biomarker collection” which each indicate the presence and/or level of a combination of biomarkers, said combination being characteristic for a discrete condition, stage of condition, subtype of condition or a prognosis for a discrete condition, stage of condition, subtype of condition.

The IGFBP2 expression level as determined in a biological sample of the subject thus allows for predicting whether said subject has, or is at risk to have, or develop a thoracic aortic aneurysm and/or thoracic aortic dissection. Hence, the IGFBP2 expression level as determined in the biological sample will also have a predictive value in evaluating whether a subject may benefit from one or more treatments aiming to slow down or inhibit thoracic aortic aneurysm and/or thoracic aortic dissection development using medicaments known in the art. The term “predicting”, and related terms such as “prediction” or “predictive” as used herein refers to an advance declaration, indication or foretelling of a response or reaction to a therapy in a subject, preferably wherein said subject has not (yet) been treated with a therapy. For example, a prediction of sensitivity (or responsiveness or susceptibility) to for example beta blockers, angiotensin II receptor blockers, or statin medicaments in a subject may be indicated by relatively low IGFBP2 expression levels, and optionally a marked decrease in IGFBP2 expression level upon an initial administration (schedule) of one or more of the above medicaments.

For example, in certain embodiments, the extent (i.e. degree) of difference of the measured IGFBP2 expression level when compared to an earlier measurement or when compared to a measurement of a control sample may be used to estimate and/or predict the speed of thoracic aortic aneurysm development. Optionally, an extrapolation to a time point where a certain risk threshold for rupture (i.e. dissection) will be achieved by may be formulated. Similarly, when upon comparison of a biological sample obtained from the patient at an earlier timepoint with a biological sample obtained from the same patient at a later timepoint the IGFBP2 expression level is lower at the later timepoint, a conclusion of a constant, substantially constant, speed of thoracic aortic aneurysm development can be concluded.

The terms “sensitivity”, “responsiveness” or “susceptibility” may be used interchangeably herein and refer to the quality that predisposes a subject having or developing a thoracic aortic aneurysm (TAA) and/or thoracic aortic dissection (TAD) to be sensitive or reactive to a certain treatment. A subject is “sensitive”, “responsive” or “susceptible” (which terms may be used interchangeably) to a certain treatment if the subject will have a clinical benefit from the treatment.

The method subject of the invention allows for determining a certain chance, or likeliness that a subject has, or is at risk to have, or develop a thoracic aortic aneurysm and/or thoracic aortic dissection. “Determining the likeliness of ” and “predicting the likeliness of’ as used herein refers to an advance declaration, indication or foretelling of a response or reaction to a therapy in a subject, or a probability of a response or reaction to a therapy in a subject, preferably wherein said subject has not (yet) been treated with a therapy.

The method described herein aims to provide a subject and/or medical practitioner with information with respect to said subject having a thoracic aortic aneurysm, or is at risk of developing a thoracic aortic aneurysm and/or thoracic aortic dissection. Hence, it is appropriate to alternatively express the method as “a method of diagnosing”, “a method of molecular profiling”, and the likes. “Molecular profiling” broadly relates to the practice of identification of one or more individual profiles that allow for more informed and effective personalized treatment options, which can result in improved patient care and enhanced treatment outcome as is known to a skilled person. However, the aspect of diagnosis may also be part of a method of treatment.

The IGFBP2 marker, peptide, polypeptide, protein or nucleic acid is “detected”, “measured”, and/or “measured” in a biological sample when the presence or absence, quantity and/or activity of said marker, peptide, polypeptide, protein, or nucleic acid is determined or measured in the biological sample, preferably substantially to the exclusion of other markers, peptides, polypeptides, proteins, or nucleic acids.

As used herein, the term “aneurysm” refers to a bulging, weak area in the wall of a blood vessel (i.e. a localised ballooning of a blood vessel). An aneurysm can be categorized by its location, shape, and cause. For example, an aneurysm may be found in many areas of the body, such as brain (cerebral aneurysm), aorta (aortic aneurysm), neck, intestines, kidney, spleen, legs. Aortic aneurysms are formed by a thinning medial layer and deterioration of the elastic lamina of the aorta wall, which will lead to a weakening of the tensile strength thereof. Aortic aneurysms are commonly identified in the thoracic and infrarenal aorta, with the latter referred to as abdominal aortic aneurysms (AAA). Both thoracic and abdominal aortic disease are characterized on cellular level by proteolytic elastic tissue degeneration and smooth muscle cell loss. A thoracic aortic aneurysm (TAA) is an aortic aneurysm that presents primarily in the thorax. Thoracic aortic aneurysms may involve the aortic root aorta, the ascending aorta, the aortic arch, the descending aorta, the thoracoabdominal aorta, or a combination thereof. It should be further appreciated that when reference is made to a risk of developing thoracic aortic aneurysm in the present specification, a risk of developing a thoracic aortic dissection/rupture is equally envisaged.

In addition, the term “aneurysm” used herein encompasses the different forms of aneurysms known to a skilled person, including “true aneurysms”, i.e. enlargements of the inner lumen caused by vessel wall expansion; “false aneurysms”, i.e. an enlargement of the lumen caused by perforation of all parts of the vessel wall forming an outer sack in communication with the inner lumen of the aorta; “localised aneurysm” or “circumscript aneurysm” which only involves portions of the aorta; and “diffuse aneurysm”, i.e. an enlargement of the ascending aorta, the aortic arch, the descending thoracic (thoracic aortic aneurysms) or abdominal aorta (abdominal aortic aneurysm), or the whole aorta (as defined by Erbel and Eggebrecht, Heart, 2006). Similarly, the aneurysm may be a “root aneurysm”, “ascending aneurysm”, “fusiform aneurysm”, or any combination thereof which each have been classified in the art as subtypes of aneurysm (e.g. in Pinard et al., Circ Res, 2019). Trauma-induced aneurysms, i.e. aneurysms caused by injury, are also envisaged by the present disclosure.

An aneurysm of a blood vessel may be generally defined as an increased outer blood vessel diameter of more than 50% of the normal diameter of a healthy individual, based on gender, body surface area and age normal values. A normal diameter of the adult thoracic aorta is known to be from about 2 to about 3 cm. A skilled person appreciates that the normal aortic diameter range depends on different subject parameters such as but not limited to age, height, body habitus, gender, and ethnicity (Paruchuri et al., Cardiology, 2015). A thoracic aorta with a diameter of more than 4.0 or 4.5 cm (i.e. a 50% increase from 2-3 cm) is considered as an aortic aneurysm. It has been estimated that the risk for rupture or dissection of a thoracic aortic aneurysm increases considerably upon reaching an aortic diameter of higher than about 5 cm, with the risk further increasing in larger aortic diameters . It is generally accepted that a thoracic aortic aneurysm with a diameter between 5 to 6 cm should be considered for clinical intervention. In yet larger thoracic aortic aneurysms (6-7 cm in diameter) or very large thoracic aortic aneurysms (diameter >7 cm) the need for intervention is typically considered urgent and emergent respectively. In specific aortic aneurysm conditions, the aortic diameter threshold for surgery might be lower (Saliba and Sia, Int J Cardiol Heart Vase, 2015). Hence, in certain embodiments the method comprises, in addition to determining the expression level of IGFBP2, determining the diameter of the thoracic aorta or a portion thereof, preferably by an imaging technique. More preferably, the imaging technique is selected from the group consisting of: positron emission tomography (PET), echocardiography, computerised tomography (CT)-scanning, Magnetic Resonance Imaging (MRI), or any combination thereof.

Related to the above, the term “thoracic aortic dissection” (TAD) refers to an injury of the thoracic aorta wherein distinct layers of the aorta are separated and blood (flow) occurs between said layers. A new lumen (false lumen) is created, which is typically accompanied by an acute drop in systemic blood pressure, potentially leading to hemopericardium (i.e. presence of blood in the pericardial sac of the heart) and a cardiac tamponade (i.e. the build-up of fluid in the pericardium resulting in a compression of the heart) having a sudden death as consequence.

In addition, a skilled person appreciates that the terms “thoracic aortic aneurysm” (TAA) and “thoracic aortic dissection” (TAD) and “thoracic aortic rupture” are conditions that do not fall under the umbrella term “heart failure”. Indeed, while TAA and TAD affect the aorta, heart failure affects the heart as such. Heart failure, often interchangeably indicated by the expression “congestive heart failure” indicates a failure of the heart to pump blood in an adequate manner to support the circulatory system. It has been described that TAA is very rarely related to heart failure and that the two conditions can be clinically clearly distinguished from each other (e.g., Jorge et al., Rev Port Cardiol (Engl Ed), 2018). Additionally, the distinction between both conditions is also apparent from for example earlier work on biomarkers for heart failure wherein BNP (natriuretic peptide B) was found to be a specific marker for heart failure and not aortic dissection (Sbarouni et al., International Journal of Cardiology, 2007).

The term “biological sample” as used herein refers and optionally abbreviated by terms such as “sample” encompasses any biological specimen obtained (i.e. isolated, removed) from a subject. Biological samples may include without limitation organ tissue (e.g. aortic wall tissue), whole blood, plasma, serum, whole blood cells, red blood cells, white blood cells (e.g. peripheral blood mononuclear cells), saliva, naso-pharyngeal fluid, oropharyngeal fluid, bronchoalveolar fluid, sputum, urine, stool (faeces), tears, sweat, sebum, nipple aspirate, ductal lavage, synovial fluid, cerebrospinal fluid, lymph, fine needle aspirates, amniotic fluid, any other bodily fluid, exudate or secretory fluid, cell lysates, cellular secretion products, and inflammation fluid.

The nature of the biological sample is not particularly limiting for the present invention. A preferred biological sample in the context of the present disclosure is a serum sample. An alternative preferred biological sample in the context of the present disclosure is a plasma sample. A skilled person is aware that a serum sample is generally described as a blood plasma sample wherein clotting factors are additionally removed. It is therefore broadly accepted that a serum sample typically comprises proteins that are not involved in blood clotting, as well as electrolytes, antibodies, antigens, hormones. Exogenous substances such as but not limited to medicaments and/or microorganisms are generally retained upon the preparation of a serum sample. Serum sample generation techniques have been described at numerous occasions in the art for combination with a plethora of detection methods and technologies and are therefore known to a skilled person (e.g. Alshammari et al., Saudi Pharm J, 2015). Preferably, the biological sample may be readily obtainable by non-invasive or minimally invasive methods. By means of illustration and not limitation, such a method may be a method of blood collection (“liquid biopsy”). Optionally, the biological sample is a blood sample that is collected, and temporarily preserved specialized containers. Another non-limiting example of such a container that is commercially available is the PAXgene Blood DNA tubes (Qiagen). The term “tissue” as used herein encompasses all types of cells of the body including cells of organs but also including blood and other body fluids recited above. The tissue may be healthy or affected by pathological alterations, e.g. inflammation or infection. The tissue may be from a living subject or may be cadaveric tissue. Preferably, the tissue is aortic wall tissue. The term “biopsy” generally refers to a sample of cells or tissues removed (extracted, isolated, and/or purified) from a living subject for examination. The weight and/or volume of the biological sample and/or reference biological sample that is obtained from a subject for analysis is not particularly limited. Without limitation, a liquid sample may have a volume between 0.1 ml and 1 ml such as 0.5 ml, or between 1 ml and 100 ml, such as 1 ml, 5 ml, 25 ml, 50 ml, 75 ml or 100 ml. A solid sample may have a weight of between 0.1 g and 20 g, such as 0.5 g, 1 g, 5 g, 7.5 g, 10 g, 15 g or 20 g.

In certain embodiments, the method may be conducted a multitude of times on a single biological sample. In certain embodiments, the IGFBP2 expression level may be determined on a multitude of biological samples obtained from the subject. In further embodiments, the multitude of samples are each considered to be serum samples, aortic wall tissue samples, or a combination thereof.

Terms such as “subject”, “patient”, and “individual” may be used interchangeably herein and refer to animals, preferably warm-blooded animals, more preferably vertebrates, and even more preferably mammals specifically including humans. Preferred subjects are human subjects including all genders and all age categories thereof. Both adult subjects, new-born subjects, and foetuses are intended to be covered by the term “subject”. Preferred subjects in the context of the invention are human subjects that are presented to a medical practitioner such as a physician with symptoms and signs indicative of an thoracic aortic aneurysm or thoracic aortic dissection. Preferred subjects in the context of the present invention are adult subjects (i.e. subject having an age of at least 18 years). Optionally, the method described herein is a method used for screening of elderly subjects, for example subjects over the age of 55 years, preferably over the age of 60 years, preferably over the age of 65 years, preferably over the age of 70 years. In certain embodiments, the subject is a subject having, or considered to have chest pain.

In preferred embodiments, the IGFBP2 is stated to be “human”, i.e. the IGFBP2 sequence may be the same as a corresponding sequence of or present in a naturally occurring human. Hence, the qualifier “human” in this connection relates to the primary sequence of the respective markers, peptides, polypeptides, proteins, or nucleic acids, rather than to its origin or source. For example, such markers, peptides, polypeptides, proteins, or nucleic acids may be present in or isolated from samples of human subjects or may be obtained by other means (e.g. by recombinant expression, cell-free transcription or translation, or non-biological nucleic acid or peptide synthesis).

An “isolated” component (such Igfbp2 protein, or IGFBP2 mRNA) refers to a component that has been substantially separated or purified away from other biological components in the cell in which the component naturally occurs, for example, extra-chromatin DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods well known to a skilled person. A skilled person readily appreciates that the term “isolated” does not require absolute purity. Instead, it denotes that such markers, peptides, polypeptides, proteins, or nucleic acids are in a discrete environment in which their abundance (conveniently expressed in terms of mass or weight or concentration) relative to other analytes is greater than in the biological sample. A discrete environment denotes a single medium, such as for example a single solution, gel, precipitate, lyophilisate, etc. Purified nucleic acids, proteins, polypeptides or peptides may be obtained by known methods including, for example, laboratory or recombinant synthesis, chromatography, preparative electrophoresis, centrifugation, precipitation, affinity purification.

The purified Igfbp2 protein or IGFBP2 mRNA may preferably constitute by weight at least about 10%, more preferably at least about 50%, such as at least about 60%, yet more preferably at least about 70%, yet more preferably at least about 80%, most preferably at least about 90% of respectively the protein content or the mRNA content of the discrete environment. Protein content may be determined, e.g. by the Lowry method (Lowry et al. J Biol Chem 1951.), optionally as described by Hartree (Anal Biochem 1972). Purity of peptides, polypeptides, or proteins may be determined by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Quantity of nucleic acids may be determined by measuring absorbance A260. Purity of nucleic acids may be determined by measuring absorbance A260/A280, or by agarose- or polyacrylamide-gel electrophoresis and ethidium bromide or similar staining.

In embodiments wherein the subject is a non-human subject such as but not limited to a horse, the present disclosure envisages determining the expression levels of corresponding genes in the non- human subject, including homologues and orthologues genes. The terms “homologue” and “orthologue” are to be interpreted according to their generally accepted meaning in the art. Hence, a skilled person appreciates that a “homologue” (interchangeably used with terms such as “homologous gene”) is a gene inherited in two species by a common ancestor. Similarly, a skilled person appreciates that an “orthologue” (interchangeably used with terms such as “orthologous gene”) is a gene in a different species that evolved from a common ancestral gene by speciation and generally retains the same function during the course of evolution. A skilled person is capable of identifying homologues and orthologues for each gene disclosed herein, or retrieve them from literature.

The method described herein is an in vitro method. “In vitro” broadly refers to outside of, or external of the body of a subject. The present of a subject is therefore not essential for performing the method described herein. Each step of the method described herein is performed without any instance of physical interaction with the body of a subject.

In certain embodiments, the level of a particular fragment of IGFBP2 is determined. Optionally, in such embodiments the method comprises measurement of a fragment of RNA transcript of IGFBP2 and/or peptide part of Igfbp2 that are part of each known isoform of the transcript or protein. In alternative embodiments, the method comprises measurement of a fragment of RNA transcript of IGFBP2 and/or peptide part of Igfbp2 that exclusively encode for or are exclusively comprised in one or more particular isoforms of IGFBP2.

Optionally, the method comprises detecting a functionally active fragment of IFGBP2. Preferably, a functional fragment retains at least about 20%, e.g. at least 30%, or at least about 40%, or at least about 50%, e.g. at least 60%, more preferably at least about 70%, more preferably at least 80%, yet more preferably at least about 85%, still more preferably at least about 90%, and most preferably at least about 95% or even about 100% or higher of the intended biological activity or functionality compared to the corresponding full length nucleic acid, protein, polypeptide or peptide. Alternatively, the method comprises detecting a functionally inactive fragment of IGFBP2 (i.e. an IGFBP2 fragment that is not capable of exerting canonical IFGBP2 functions).

The term “fragment” with reference to the IGFBP2 mRNA transcript generally refers to a 5’ - and/or 3’- truncated form of a nucleic acid, but may also indicate internal fragments. Preferably, a fragment may comprise at least about 30%, preferably at least about 50%, preferably at least about 70%, preferably at least about 80%, more preferably at least about 85%, more preferably at least about 90%, more preferably at least about 95%, most preferably 99% of the nucleic acid sequence length of the IGFBP2 transcript. For example, insofar not exceeding the length of the full-length nucleic acid, a fragment of an IGFBP2 nucleic acid sequence (i.e. transcript sequence) may include a sequence of at least 5 consecutive nucleotides, preferably at least 10 consecutive nucleotides, preferably at least 20 consecutive nucleotides, preferably at least 30 consecutive nucleotides, preferably at least 40 consecutive nucleotides, such as for example about 50 consecutive nucleotides, preferably about 60, preferably about 70, preferably about 80, preferably about 90, preferably about 100, preferably about 200, preferably about 300, preferably about 400, preferably about 500, preferably about 600, preferably about 700, preferably about 800, preferably about 900, preferably about 1000, preferably about 1100, preferably about 1200_s preferably about 1300, preferably about 1400, or preferably about 1500 consecutive nucleotides of the corresponding full-length IGFBP2 transcript sequence, in particular IGFBP2 mRNA.

The term “fragment” as used throughout this specification with reference to Igfbp2 protein generally denotes a portion of said protein, such as typically an N- and/or C-terminally truncated form of the Igfbp2 protein, but may also indicate internal fragments. Preferably, a fragment may comprise at least about 30%, preferably at least about 50% or at least about 70%, preferably at least about 80%, more preferably at least about 85%, more preferably at least about 90%, more preferably at least about 95%, most preferably 99% of the amino acid sequence length of said peptide, polypeptide, or protein. For example, insofar not exceeding the length of the full-length peptide, polypeptide, or protein, a fragment may include a sequence of at least 5 consecutive amino acids, preferably at least 10 consecutive amino acids, preferably at least 20 consecutive amino acids, preferably at least 30 consecutive amino acids, preferably at least 40 consecutive amino acids, such as for example about 50 consecutive amino acids, preferably about 60, preferably about 70, preferably about 80, preferably about 90, preferably about 100, preferably about 200, most preferably about 300, Igfbp2 protein sequence.

The term “fragment” encompasses fragments arising by any mechanism, in vivo and/or in vitro, such as, without limitation, by alternative transcription or translation, exo- and/or endo-proteolysis, exo- and/or endo-nucleolysis, or degradation of the peptide, polypeptide, protein, or nucleic acid, such as, for example, by physical, chemical and/or enzymatic proteolysis or nucleolysis.

Optionally, the method comprises detection of the level of IGFBP2 expression in a biological sample and determining whether said IGFBP2 level is altered when compared to a control sample. A skilled person appreciates that the term “altered” indicates a change that is considered significant by means of either direct comparison of the measured value with the value provided by the control sample, or is considered significant when compared to the value provided by the control sample by means of any suitable statistical analysis or test.

Hence, a differential expression level of IGFBP2 in the biological sample when compared to the control sample is indicative for the presence or development of thoracic aortic aneurysm and/or dissection in a subject. Terms such as “different level” and “differential expression level” imply a measurable difference in expression level (i.e. the extent to which an analyte is present in an analysed sample and therefore a proxy for the extent to which said analyte is present in the subject from which said sample is obtained from), and evidently implies statistical significance of the difference between the expression level of the biological sample and the reference biological sample, or at least a trend that may be deducted upon analysing the expression levels. By means of illustration and not limitation, a suitable threshold for attributing statistical significance in expression level is a 21og change characterised by a p value <0.001 and a false discovery rate (FDR) <0.05.

Alternatively, the “different (expression) level” may be expressed as a “deviation of expression level” of one or more of the analysed biomarkers in a sample (in the context of the present invention obtained from a subject suspected of having presence or development of thoracic aortic aneurysm and/or dissection or a subject being screened for the presence of a thoracic aortic aneurysm and/or dissection) when compared to a reference biological sample (e.g. a healthy subject known to not have said aortic aneurysm and/or dissection) or internal standard. A “deviation” of a first expression level of a biomarker in the sample obtained from the subject from a second expression level of said biomarker in the internal standard may generally encompass any direction (e.g. increase: first value > second value; or decrease: first value < second value) and any extent of alteration.

For example, a deviation may encompass a decrease of a first value by, without limitation, at least about 10% (about 1. 1-fold or more), or by at least about 20% (about 1.2-fold or more), or by at least about 30% (about 1.3-fold or more), or by at least about 40% (about 1.4-fold or more), or by at least about

50% (about 1.5-fold or more), or by at least about 60% (about 1.6-fold or more), or by at least about

70% (about 1.7-fold or more), or by at least about 80% (about 1.8-fold or more), or by at least about

90% (about 1.9-fold or more), or by at least about 100% (about 2-fold or more), or by at least about

150% (about 2.5 -fold or more), or by at least about 200% (about 3 -fold or more), or by at least about 500% (about 6-fold or more), or by at least about 700% (about 8-fold or more), or like, relative to a second value with which a comparison is being made.

For example, a deviation may encompass an increase of a first value by, without limitation, at least about 10% (about 1.1 -fold or more), or by at least about 20% (about 1.2-fold or more), or by at least about 30% (about 1.3-fold or more), or by at least about 40% (about 1.4-fold or more), or by at least about 50% (about 1.5-fold or more), or by at least about 60% (about 1.6-fold or more), or by at least about 70% (about 1.7-fold or more), or by at least about 80% (about 1.8-fold or more), or by at least about 90% (about 1.9-fold or more), or by at least about 100% (about 2-fold or more), or by at least about 150% (about 2.5-fold or more), or by at least about 200% (about 3-fold or more), or by at least about 500% (about 6-fold or more), or by at least about 700% (about 8-fold or more), or like, relative to a second value with which a comparison is being made.

In the context of the present invention, a deviation refers to a statistically significant observed alteration in the expression level of IGFBP2 in the method subject of the present invention. For example, a deviation may refer to an observed alteration or increase, which falls outside of error margins of reference levels obtained from an internal standard and/or a reference biological sample (as expressed, for example, by standard deviation (SD) or standard error (SE), or by a predetermined multiple thereof, e.g. ±lxSD or ±2xSD or ±3xSD, or ±lxSE or ±2xSE or ±3xSE). Deviation or reduction may also refer to a value falling outside of a reference range defined by the expression levels measured in multiple reference biological samples (for example, outside of a range which comprises >40%, >50%, >60% ,>70%, >75%, >80%, >85%, >90%, >95%, or even >100% of expression levels measured in said reference biological samples). Alternatively, a deviation may be concluded if an observed alteration is beyond a given threshold or cut-off. Such threshold or cut-off may be selected as generally known in the art to provide for a chosen sensitivity and/or specificity of the prediction methods, e.g. sensitivity and/or specificity of at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%.

“Control sample” or alternatively “reference sample” as used herein refers to a biological sample obtained from a healthy subject. It is evident that a control sample has the same tissue or cellular origin as the sample under investigation. For example, when the biological sample used for the method described herein is a serum sample from a subject under investigation, a suitable control sample is a serum sample from a healthy subject. It is appreciated that determining whether a subject is “healthy” with respect to thoracic aortic aneurysm and/or dissection is well within the capabilities of a skilled person. Said earlier determination may be based on the method according to the invention that was conducted on the (healthy) subject at an earlier point in time, and/or on imaging data obtained for said (healthy) subject.

Optionally, the expression level of a control sample (hereafter interchangeably indicated by terms such as but not limited to “reference (expression) level” and “reference (expression) value”) may be the representative IGFBP2 expression level obtained from at least two control samples. Optionally, the reference expression level may be the average or the mean IGFBP2 expression level obtained from at least two control samples.

Optionally, the reference expression level or reference expression value may be derived from a digital database, such as a computer database. Said database may comprise a collection of (control) biological samples wherein the level of IGFBP2 has been determined. Alternatively, the database may generate a suitable reference expression level upon input by a user of one or more subject characteristics, such as age, gender, and/or ethnicity.

Optionally, the reference expression level or reference expression value may be derived from an internal standard which is added to the biological sample of the subject under investigation. The internal standard may be any reference IGFBP2 counterpart that allows for calibration of the method, detection of IGFBP2, and/or quantification of IGFBP2. A skilled person appreciates that suitable reference biomarker counterparts are subject of change in function of the detection means that are used for measuring a given biomarker, in the present context IGFB2. By means of illustration and not limitation, the internal standard may therefore comprise one or more stable isotope labelled reference Igfbp2 protein, peptide, or collection of peptides.

In certain embodiments, the subject is considered to have, or is considered of being in the process of developing thoracic aortic aneurysm and/or thoracic aortic dissection when the IGFBP2 expression is at least about 10% increased when compared to the IGFBP2 expression in the control sample. In preferred embodiments, the IGFBP2 expression is at least about 25% increased, preferably at least about 35% increased, preferably at least about 50% increased, more preferably at least about 75% increased, most preferably at least about 100% increased when compared to the IGFBP2 expression in the control sample. It is to be understood that the expression “at least x%” effectively indicates a range of “from x% to infinity”.

In certain embodiments, the subject is considered to have, or is considered of being in the process of developing thoracic aortic aneurysm and/or thoracic aortic dissection when the IGFBP2 expression is at least about 1.25 fold the IGFBP2 expression in the control sample. In preferred embodiments, the IGFBP2 expression is at least about 1 .5 fold, preferably at least about 2 fold, preferably at least about 2.5 fold, more preferably at least about 5 fold, most preferably at least about 10 fold when compared to the IGFBP2 expression in the control sample.

Generally, it can be considered that higher expression levels of IGFBP2 are indicative for an increase in severity and/or pace of development of thoracic aortic aneurysm and/or thoracic aortic dissection.

In particular embodiments, the expression level of IGFBP2 is indicative for the severity of the thoracic aortic aneurysm and/or thoracic aortic dissection. By means of illustration and not limitation, in such embodiments a subject having a relatively larger significant increase in IGFBP2 expression level when compared to the reference expression level is considered to have a more severe thoracic aortic aneurysm and/or thoracic aortic dissection when compared to a subject having a relatively smaller significant increase in IGFBP2 expression level when compared to the reference expression level.

In particular embodiments, the expression level of IGFBP2 is indicative for the speed of thoracic aortic aneurysm and/or thoracic aortic dissection development. By means of illustration and not limitation, in such embodiments a subject having a relatively larger significant increase in IGFBP2 expression level when compared to the reference expression level is considered to develop thoracic aortic aneurysm or thoracic aortic dissection faster (i.e. at a higher pace) when compared to a subject having a relatively smaller significant increase in IGFBP2 expression level when compared to the reference expression level. In further embodiments, the expression level of IGFBP2 may be measured at multiple points in time to evaluate thoracic aortic aneurysm disease progression, or absence of disease progression. In such embodiments, a further increase in IGFBP2 expression level upon comparison of a first point in time (i.e. earlier timepoint) and a second point in time (i.e. later timepoint) may indicate that disease progression is accelerating. Contrarily, a decrease in IGFBP2 expression level upon comparison of a first point in time (i.e. earlier timepoint) and a second point in time (i.e. later timepoint) may indicate that disease progression is slowing down or halting, for example as a consequence of medical intervention (e.g. medicinal intervention and/or surgical intervention). The method described in the present disclosure thus provides a tool for risk-stratification of patients into “fast” and “slow” progression, and optionally further sub groups of thoracic aortic aneurysm progression. Such a stratification allows for further personalized and effective treatment strategies.

Optionally, the control sample is obtained from a subject characterized by normal aortic diameters, “normal aortic diameters” are considered aortic diameters that are within the boundaries of diameters that are commonly observed in subjects as detailed above.

In certain embodiments, the method is a method of detecting asymptomatic thoracic aortic aneurysm, asymptomatic thoracic aortic dissection, and/or symptomatic thoracic aortic dissection. In certain embodiments, the method is a method of detecting asymptomatic thoracic aortic aneurysm. “Asymptomatic” is to be considered throughout the present disclosure in accordance with the generally accepted meaning in the art, i.e. the complete absence symptoms to a medical practitioner. An asymptomatic subject is therefore a subject that is not marked by, or presenting with signs or symptoms of a disease, in the present context thoracic aortic aneurysm. In further embodiments, the subject is an asymptomatic subject to which the method subject of the invention is applied in a screening context (asymptomatic screening), or a routine medical examination such as but not limited to a routine (optionally periodic) blood analysis.

In certain embodiments, the method is a method of detecting symptomatic thoracic aortic dissection. Optionally the subject that is tested for the presence of symptomatic thoracic aortic dissection is characterized by one or more symptoms selected from the group consisting of: (sudden) severe chest pain, (sudden) severe back pain, (sudden) severe stomach pain, loss of consciousness, shortness of breath, stroke-like symptoms, sudden vision impairment, sudden speech impairment, partial paralysis, attenuated pulse in a limb when compared to the other corresponding limb, leg pain, difficulty of walking. In the context of thoracic aortic dissection, the sudden severe chest pains and sudden upper back pain are commonly described in the art as ripping sensations and/or tearing sensations.

Optionally, the subject has an increased risk and/or underlying risk to develop thoracic aortic aneurysm and/or thoracic aortic dissection. Known risk factors include without limitation aging, atherosclerosis, blunt chest trauma, elevated blood pressure (i.e. hypertension), bicuspid aortic valve, coarctation (narrowing) of the aorta, connective tissue disorders (such as Marfan syndrome and Ehlers-Danlos syndrome) and rare genetic disorders, heart surgery, pregnancy, arteritis, syphilis, substance abuse (e.g. cocaine or methamphetamine abuse), periodical weight lifting (e.g. bodybuilding), smoking, diabetes, medication use, and hypercholesterolemia. In particular embodiments, the method comprises an additional step of applying a normalization coefficient on the IGFBP2 expression level based on the occurrence of one or more of the above-mentioned risk factors.

In certain embodiments, the method described herein is a method for monitoring clinical progression of thoracic aortic aneurysm in a subject wherein said thoracic aortic aneurysm is detected in sample obtained from said subject on at least one earlier point in time. Optionally, the IGFBP2 expression levels of two or more different time points is detected and compared at the different time points.

The particular time interval between measurements for applications such as monitoring of disease progression is not particularly limiting for the invention and may therefore be an interval of at least about 1 year, preferably at least about 6 months, preferably at least about 3 months, preferably at least about 1 month, more preferably at least about 2 weeks, most preferably at least about 1 week.

In certain embodiments, a decrease in IGFBP2 expression at the later time point compared to the earlier time point (i.e. an IGFBP2 upregulation) indicates a favourable clinical progression of the thoracic aortic aneurysm in the subject, whereas an increase in IGFBP2 expression at the later time point compared to the earlier time point (i.e. an IGFBP2 downregulation) indicates progression of the subject towards greater severity of the thoracic aortic aneurysm, such as wherein the subject has progressed to a severe thoracic aortic aneurysm that requires, medication, hospitalization, intensive care treatment, extension of hospitalization, or extension of the intensive care treatment, or wherein the subject is considered to have a concrete risk of experiencing thoracic aortic dissection. Such upregulation or downregulation of IGFBP2 expression levels between the different time points may be assessed by comparing the intra-patient measurements obtained at the different time points directly, or alternatively by comparing each of the measurements to a suitable reference value (optionally directly derived from a control sample) as described above.

In a context of disease progression monitoring, certain threshold IGFBP2 expression levels may optionally be linked to the initiation of a certain treatment. In further embodiments certain threshold IGFBP2 expression levels may be linked to the initiation of a particular treatment with one or more pharmaceutically active ingredients know in the art, such as those described further below. In yet further embodiments, certain threshold IGFBP2 expression levels may be linked to specific dosages of said one or more pharmaceutically active ingredient that are to be used. In further embodiments wherein the subject is treated with a medicament (i.e. a pharmaceutically active ingredient), certain IGFBP2 expression levels may be linked to treatment regimens of one or more pharmaceutically active ingredients that are to be used.

Additionally, the method described herein is suitable to predict or monitor an outcome of thoracic aortic aneurysm treatment in a subject. The term “outcome” generally refers to the evaluation undertaken to assess the results or consequences of management and procedures (i.e. the interventions) used in combatting a disease in order to determine the efficacy, effectiveness, safety, practicability, etc., of these interventions, e.g. in individual cases or series. The term “monitoring outcome” broadly refers to a process of assessing (i.e. monitoring) the consequences of treating a subject for a thoracic aortic aneurysm which the subject is determined to have based on the IGFBP2 expression level above a certain threshold level or above the IGFBP2 expression level in a control sample, and monitoring whether said individual is responding or not to the treatment. The term “predicting outcome” broadly refers to a process of predicting the consequences of treating a subject for a thoracic aortic aneurysm which the subject is determined to have based on the IGFBP2 expression level above a certain threshold level or above the IGFBP2 expression level in a control sample, and predicting whether said individual is likely to respond or not to the treatment.

The terms “treat” or “treatment” encompass the therapeutic treatment of an already developed disease or condition, such as the therapy of both an already developed clinical image indicative for thoracic aortic aneurysm or an anticipated clinical image indicative for a thoracic aortic aneurysm (i.e. a clinical image that is expected to occur in a future point in time, such as thoracic aortic dissection). Beneficial or desired clinical results may include, without limitation, alleviation of one or more symptoms or one or more biological markers, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and the like. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

In certain embodiments, the method described herein is a method for predicting (the occurrence of) thoracic aortic dissection or rupture in a subject. In such embodiments, the method may predict the likelihood that thoracic aortic dissection or rupture will occur. In yet further embodiments, the method may predict the likelihood that thoracic aortic dissection or rupture will occur within a certain time window (i.e. time interval, time frame). The subject may be a subject wherein thoracic aortic aneurysm was diagnosed at an earlier point in time.

For example, the method may predict a likelihood of at least about 5%, preferably at least about 10%, preferably at least about 20%, preferably at least about 30%, preferably at least about 40%, preferably at least about 50%, preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 85%, more preferably at least about 90%, most preferably at least about 95% that aortic dissection or rupture will occur in the subject.

Alternatively, the method may predict that aortic dissection or rupture will occur in the subject within about 1 year, preferably within about 6 months, preferably within about 3 months, preferably within about 1 month, preferably within about 2 weeks, preferably within about 1 week, more preferably within about 5 days, more preferably within about 2 days, more preferably within about 1 day, more preferably within about 12 hours, more preferably within about 6 hours, most preferably within about 2 hours.

Yet alternatively, the method may predict that a thoracic aortic dissection or rupture will occur with a likelihood of at least about 5% within about 1 year, preferably at least about 10% within about 6 months, preferably at least about 20% within about 3 months, preferably at least about 30% within about 1 month, preferably at least about 40% within about 2 weeks, preferably at least about 50% within about 1 week, preferably at least about 60% within about 5 days, more preferably at least about 70% within about 2 days, more preferably at least about 80% within about 1 day, more preferably at least about 85% within about 12 hours, more preferably at least about 90% within about 6 hours, most preferably at least about 95% within about 2 hours.

The subject may be a subject that has a genetic predisposition to develop thoracic aortic aneurysm, or a genetic predisposition which increases the chances to develop thoracic aortic aneurysm. Genetic risk factors contributing to or responsible for TAA development have been described in the art (e.g. in Pinard et al., Circ Res, 2019) and are therefore known to a person skilled in the art. Such subjects may interchangeably be indicated as subjects having a heritable risk for thoracic aortic diseases (i.e. aneurysms and dissections) (HTAD).

In certain embodiments, the subject is a subject having a genetic impairment (i.e. a mutation which negatively affect the normal function of a gene) in one or more genes selected from the group consisting of: smooth muscle actin al (ACTA2), procollagen type III al (COL3A1), fibrillin-1 (FBN1), smooth muscle myosin heavy chain 11 (MYH11), mothers against decapentaplegic drosophila homolog 3 (SMAD3), TGF-P2 (TGFB2), TGF-P receptor type I (TGFBR1), TGF-P receptor type II (TGFBR2), myosin light chain kinase (MYLK), lysyl oxidase (LOX), protein kinase cGMP -dependent type 1 (PRKG1), EGF containing fibulin-like extracellular matrix protein 2 (fibulin-4) (EFEMP2), elastin (ELN), fibrillin 2 (FBN2), filamin A (FLNA), notch 1 (NOTCH1), solute carrier family 2 member 10 (SLC2A10), mothers against decapentaplegic drosophila homolog 4 (SMAD4), mothers against decapentaplegic drosophila homolog 6 (SMAD6), elastin microfibril interfacer 1 (EMILIN 1), a disintegrin and metalloproteinase with thrombospondin motifs-like protein 6 (THSD4), testin (TES), procollagenlysine, 2-oxoglutarate 5 -dioxygenase 1 (PLOD1), procollagenlysine, 2-oxoglutarate 5- dioxygenase 3 (PLOD3), FKBP prolylisomerase 14 (FKBP14), collagen type 5 alpha-1 chain (COL5A1), collagen type 5 alpha-2 chain (COL5A2), collagen type 1 alpha-1 chain (COL1A1), collagen type 1 alpha-2 chain (COL1A2), ABL proto-oncogene 1 (ABL1), jagged canonical notch ligand 1 (JAG1), histone acetyltransferase P300 (EP300), calcium-activated potassium channel subunit alpha 1 (KCNMA1), polycystin 1 (PKD1), polycystin 2 (PKD2), SKI protooncogene (SKI), biglycan (BGN), forkhead box E3 (FOXE3), hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4), methionine adenosyltransferase II a (MAT2A), microfibrillar-associated protein 5 (MFAP5), mothers against decapentaplegic drosophila homolog 2 (SMAD2), TGF-P3 (TGFB3), latent TGF-P3 binding protein 3 (LTBP3), ariadne drosophila homolog 1 (ARIH1), and importin 8 (IPO8) (Rodrigues Bento et al., Annu Rev Genomics Hum Genet, 2022).

A skilled person appreciates that genetic impairment indicates the presence of at least one mutation which negatively affects the normal function of a gene. The exact mutation in the genes mentioned herein is not particularly limiting for the invention. Hence, the mutation may be a substitution (i.e. a missense mutation or a nonsense mutation), an insertion, a deletion, a duplication, an inversion, a frameshift mutation, or a repeat expansion. In embodiments wherein more than one mutation is present said mutations may be any combination of substitutions (missense mutations, nonsense mutations), insertions, deletions, duplications, inversions, frameshift mutations, and repeat expansions. Further genetic alterations that may lead to reduced or abolished expression or function of any gene equally encompass large-scale mutations, such as copy number aberrations (CNA) and chromosomal aberrations. The term “deletion” as used herein with regard to genetic alterations refers to a mutation wherein one or more nucleotides, typically consecutive nucleotides, of a nucleic acid are removed, (i.e. deleted) from the nucleic acid. The term “insertion” as used herein with regard to genetic alterations refers to a mutation wherein one or more nucleotides, typically consecutive nucleotides, are added (i.e. inserted) into a nucleic acid. The term “substitution” as used herein with regard to genetic alterations refers to a mutation wherein one or more nucleotides of a nucleic acid are each independently replaced (i.e. substituted) by another nucleotide.

In preferred embodiments, the subject is a subject having a genetic impairment in one or more genes selected from the group consisting of: smooth muscle actin a2 (ACTA2), procollagen type III al (COL3A1), fibrillin-1 (FBN1), smooth muscle myosin heavy chain 11 (MYH11), mothers against decapentaplegic drosophila homolog 3 (SMAD3), TGF-P2 (TGFB2), TGF-P receptor type I (TGFBR1), TGF-P receptor type II (TGFBR2), myosin light chain kinase (MYLK), lysyl oxidase (LOX), protein kinase cGMP -dependent type 1 (PRKG1), EGF containing fibulin-like extracellular matrix protein 2 (fibulin-4) (EFEMP2), and importin 8 (IPO8).

In further preferred embodiments, the subject is a subject having a genetic impairment in one or more genes selected from the group consisting of: smooth muscle actin a2 (ACTA2), procollagen type III al (COL3A1), fibrillin-1 (FBN1), smooth muscle myosin heavy chain 11 (MYH11), mothers against decapentaplegic drosophila homolog 3 (SMAD3), TGF-P2 (TGFB2), TGF-P receptor type I (TGFBR1), TGF-P receptor type II (TGFBR2), myosin light chain kinase (MYLK), and importin 8 (IPO8).

In certain embodiments the subject has an autosomal dominant heritable risk for TAD. In such embodiments, the subject may have a genetic impairment in one or more genes selected from the group consisting of: smooth muscle actin a2 (ACTA2), procollagen type III al (COL3A1), fibrillin-1 (FBN1), smooth muscle myosin heavy chain 11 (MYH11), mothers against decapentaplegic drosophila homolog 3 (SMAD3), TGF-P2 (TGFB2), TGF-P receptor type I (TGFBR1), TGF-P receptor type II (TGFBR2), myosin light chain kinase (MYLK), lysyl oxidase (LOX), protein kinase cGMP -dependent type 1 (PRKG1), elastin (ELN), fibrillin 2 (FBN2), notch 1 (NOTCHl), mothers against decapentaplegic drosophila homolog 4 (SMAD4), mothers against decapentaplegic drosophila homolog 6 (SMAD6), elastin microfibril interfacer 1 (EMILIN1), a disintegrin and metalloproteinase with thrombospondin motifs-like protein 6 (THSD4), testin (TES), FKBP prolylisomerase 14 (FKBP14), collagen type 5 alpha-1 chain (COL5A1), collagen type 5 alpha-2 chain (COL5A2), collagen type 1 alpha-1 chain (COL1A1), collagen type 1 alpha-2 chain (COL1A2), ABL proto-oncogene 1 (ABL1), jagged canonical notch ligand 1 (JAG1), histone acetyltransferase P300 (EP300), calcium-activated potassium channel subunit alpha 1 (KCNMA1), polycystin 1 (PKD1), polycystin 2 (PKD2), SKI protooncogene (SKI), forkhead box E3 (FOXE3), hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4), methionine adenosyltransferase II a (MAT2A), microfibrillar-associated protein 5 (MFAP5), mothers against decapentaplegic drosophila homolog 2 (SMAD2), TGF-P3 (TGFB3), and ariadne drosophila homolog 1 (ARIH1). In alternative embodiments the subject has an autosomal recessive heritable risk for TAD. In such embodiments, the subject may have a genetic impairment in one or more genes selected from the group consisting of: EGF containing fibulin-like extracellular matrix protein 2 (fibulin-4) (EFEMP2), solute carrier family 2 member 10 (SLC2A10), latent TGF-P3 binding protein 3 (LTBP3), procollagenlysine, 2 -oxoglutarate 5-dioxygenase 1 (PLOD1), procollagenlysine, 2-oxoglutarate 5-dioxygenase 3 (PLOD3), and importin 8 (IPO8).

In yet alternative embodiments the subject has an X-linked recessive heritable risk for TAD. In such embodiments, the subject may have a genetic impairment in fdamin A (FLNA), biglycan (BGN), or both.

In yet alternative embodiments the subject has a genetic impairment in one or more genes selected from the group consisting of: fibrillin-1 (FBN1), importin 8 (IPO8), and mothers against decapentaplegic drosophila homolog 3 (SMAD3).

In certain embodiments, the subject is a subject that is diagnosed to have or considered to have a syndrome selected from the group consisting of: smooth muscle dysfunction syndrome, (vascular or classical) Ehlers-Danlos syndrome (optionally kyphoscoliotic type 1 or 2, or arthrochalasia type), Alagille syndrome, Rubinstein-Taybi syndrome, Liang-Wang syndrome, Polycystic Kidney Disease, Marfan syndrome or musculoskeletal manifestations thereof, Loeys-Dietz syndrome 3, Loeys-Dietz syndrome 4, Loeys-Dietz syndrome 1, Loeys-Dietz syndrome 2, cutis laxa syndrome such as cutis laxa type IB syndrome, congenital contractual arachnodactyly, cardiac valvular dysplasia, arterial tortuosity syndrome, juvenile polyposis/hereditary hemorrhagic telangiectasia syndrome, Shprintzen-Goldberg syndrome, Loeys-Dietz syndrome 5, and dental anomalies and short stature syndrome.

The genetic impairment may be identified in the subject providing the biological sample for the method described herein, but may equally have been identified in a blood relative subject, i.e. subjects that share a common ancestor such as but not limited to one or both parents, grandparents, siblings, and/or one or more children. Further envisaged are (large scale) screening programs relying on the method described herein.

The subject may be a subject that has a normal aortic valve morphology (i.e. structural architecture). Such subjects are considered to have a tricuspid aortic valve, i.e. an aortic valve characterized by three cusps or leaflets. Alternatively, the subject is a subject characterized by the presence of a bicuspid aortic valve.

As used herein, the term “bicuspid aortic valve”, abbreviated as “BAV” refers to an aortic valve autonomy that approximately occurs in up to 2% of the general population (Tessier et al., Aorta (Stamford), 2021). It is known by those of skill in the art that a bicuspid aortic valve is a cardiac congenital aberration (i.e. anomaly), wherein two of the aortic valvular leaflets fuse, resulting in a valve that is “bicuspid”, in contrast to the normal “tricuspid” aortic valve. As used herein the term bicuspid aortic valve refers to any anatomical configuration in which two cusps are fused, irrespectively of the type of fusion. The occurrence of bicuspid aortic valve is often associated with other congenital cardiac lesions. The most frequent associated finding is dilation of the proximal ascending aorta secondary to abnormalities of the aortic media. Changes in the aortic media are present independent of whether the valve exerts a normal function, stenotic function, or incompetent function. In view hereof, a bicuspid aortic valve is generally considered to be a condition affecting both the valve and the aorta, including the ascending aorta, aortic arch, descending aorta, and abdominal aorta. A bicuspid aortic valve is further associated with the occurrence of cardiac events and even cardiac events that have a high mortality rate, such as ascending aortic aneurysm and dissection, and significant valvular dysfunction. Non-limiting examples of valvular dysfunction include aortic stenosis and aortic insufficiency.

In certain embodiments, the subject is a subject having idiopathic degenerative disease. In further embodiments, the subject is a subject diagnosed to have or considered to have idiopathic thoracic aortic aneurysm. “Idiopathic disease” as used herein refers to a disease with unknown cause or mechanism, i.e. a disease that has an impression of occurring spontaneously without a clear reason or cause. In the art, idiopathic may be alternatively indicated by the terms “essential”, “primary”, or less common “agnogenic”, and situational “cryptogenic”. Typically a subject is diagnosed to have an idiopathic disease or disorder by exclusion of other non-idiopathic diseases displaying a similar clinical manifestation. This process is commonly referred to as “diagnosis of exclusion” or “diagnosis by exclusion”. Idiopathic degenerative aneurysms have been discussed in the art (e.g. Krisch et al, Asian Cardiovasc Thorac Ann, 2006) and the condition is therefore well known to a skilled person.

In certain embodiments, the method described herein further comprises detection of one or more additional biomarkers in the biological sample of the subject in addition to IGFBP2, preferably wherein the one or more additional biomarker is selected from the group consisting of: smooth muscle actin a2 (ACTA2), procollagen type III al (COL3A1), fibrillin-1 (FBN1), smooth muscle myosin heavy chain 11 (MYH11), mothers against decapentaplegic drosophila homolog 3 (SMAD3), TGF-P2 (TGFB2), TGF-P receptor type I (TGFBR1), TGF-P receptor type II (TGFBR2), myosin light chain kinase (MYLK), lysyl oxidase (LOX), protein kinase cGMP-dependent type 1 (PRKG1), EGF containing fibulin-like extracellular matrix protein 2 (fibulin-4) (EFEMP2), elastin (ELN), fibrillin 2 (FBN2), filamin A (FLNA), notch 1 (NOTCHl), solute carrier family 2 member 10 (SLC2A10), mothers against decapentaplegic drosophila homolog 4 (SMAD4), mothers against decapentaplegic drosophila homolog 6 (SMAD6), elastin microfibril interfacer 1 (EMILIN1), a disintegrin and metalloproteinase with thrombospondin motifs-like protein 6 (THSD4), testin (TES), procollagenlysine, 2-oxoglutarate 5- dioxygenase 1 (PLOD1), procollagenlysine, 2-oxoglutarate 5-dioxygenase 3 (PLOD3), FKBP prolylisomerase 14 (FKBP14), collagen type 5 alpha-1 chain (COL5A1), collagen type 5 alpha-2 chain (COL5A2), collagen type 1 alpha-1 chain (COL1A1), collagen type 1 alpha-2 chain (COL1A2), ABL proto-oncogene 1 (ABL1), jagged canonical notch ligand 1 (JAG1), histone acetyltransferase P300 (EP300), calcium-activated potassium channel subunit alpha 1 (KCNMA1), polycystin 1 (PKD1), polycystin 2 (PKD2), SKI protooncogene (SKI), biglycan (BGN), forkhead box E3 (FOXE3), hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4), methionine adenosyltransferase II a (MAT2A), microfibrillar-associated protein 5 (MFAP5), mothers against decapentaplegic drosophila homolog 2 (SMAD2), TGF-P3 (TGFB3), latent TGF-P3 binding protein 3 (LTBP3), ariadne drosophila homolog 1 (ARIH1), and importin 8 (IPO8).

In addition to the canonical gene product of the listed genes, non-canonical gene products of said genes are retrievable from the repositories mentioned above. It is appreciated by a skilled person that in this context “detection” includes but is by no means limited to measurement of expression, quantitation of expression level, detection of certain isoforms, detection of certain nucleic acid mutations and/or amino acid mutations. Hence, the method may comprise measuring the expression levels of the canonical transcript and/or protein product of said genes, a non-canonical transcript and/or protein product of said genes, or any combination thereof, in addition to IGFBP2.

The one or more additional biomarkers may relate to the aortic extracellular matrix, aortic smooth muscle cells, or TGF-P signalling. Optionally, at least one additional biomarker is detected that relates to one or more molecular mechanisms selected from the group consisting of: maintenance and/or production of elastic fibre, maintenance and/or production of collagen fibre, TGF-P signalling, maintenance and/or generation of smooth muscle cells.

The one or more additional biomarkers that are detected may relate to a pathway selected from the group consisting of: hepatic fibrosis/hepatic stellate cell activation, adipogenesis pathway, role of macrophages fibroblasts and endothelial cells in rheumatoid arthritis, axonal guidance signalling, integrin signalling, p53 signalling, signalling by Rho family GTPases, leukocyte extravasation signalling, calcium signalling, ILK signalling, clathrin-mediated endocytosis signalling, ephrin receptor signalling, RhoA signalling, regulation of actin-based motility by Rho, complement system, Wnt/Ca + pathway, HMGB1 signalling, germ cell-Sertoli cell junction signalling, RhoGDI signalling, neurotrophin/TRK signalling, xenobiotic metabolism signalling, glucocorticoid receptor signalling, hypoxia signalling in the cardiovascular system, HIFla signalling, production of nitric oxide and reactive oxygen species in macrophages, EIF2 signalling, assembly of RNA polymerase I complex, IL- 8 signalling, mTOR signalling, VDR/RXR activation, LXR/RXR activation, role of osteoblasts osteoclasts and chondrocytes in rheumatoid arthritis, actin cytoskeleton signalling, atherosclerosis signalling, granulocyte adhesion and diapedesis, role of IL-17F in allergic inflammatory airway diseases, pyrimidine ribonucleotides interconversion, Wnt/p-catenin signalling, renal cell carcinoma signalling, dendritic cell maturation, PCP pathway, antigen presentation pathway, or any combination thereof.

The subject may be treated with one or more pharmaceutically active ingredients prior to conducting the method described herein, upon conducting the method described herein, and/or after conducting the method described herein. For example, the subject may receive one or more medicaments directed to treating a thoracic aortic aneurysm or dissection prior to detection of IGFBP2 in a biological sample from the subject. Preferably, the biological sample is obtained from said subject 1 hour, 2 hours, 4 hours 8 hours, 12 hours, 24 hours, or more than 24 hours after administration of the medicament to the subject. Alternatively, the biological sample wherein the IGFBP2 expression level is determined may have been obtained from a subject substantially simultaneously with the point in time wherein one or more medicaments directed to treating a thoracic aortic aneurysm or dissection are administered. By means of illustration and not limitation, the substantially simultaneous obtainment of the biological sample at treatment time may be initiated by a skilled practitioner in situations of medical emergency (e.g. a subject entering the medical emergency compartment complaining of heavy chest pains). Yet alternatively, the medicament may be administered to the subject after IGFBP2 expression levels are determined in a biological sample derived from said subject. In such embodiments, medicaments are administered to a subject after determination that the subject is indeed suffering from, or developing, a thoracic aortic aneurysm or dissection. In such embodiments, a prediction of efficacy and/or indication of suitability of treatment by a medicament is obtained by determining IGFBP2 expression levels in an initial step of the method, and a predicted suitable, or even predicted efficacious medicament is administered to the subject in a subsequent step of the method.

Suitable groups of pharmaceutically active ingredients include without limitation beta blockers ( - blockers), angiotensin II receptor blockers, and statins (HMG-CoA reductase inhibitors). In embodiments wherein the subject is using a beta blocker or is ordered to start using a beta blocker, both nonselective beta blockers, i selective beta blockers, 2 selective beta blockers, 3 selective beta blockers are envisaged.

By means of illustration and not limitation, suitable non selective beta blockers include Propranolol, Bucindolol, Carteolol, Carvedilol, Labetalol, Nadolol, Oxprenolol, Penbutolol, Pindolol, Sotalol, and Timolol. By means of illustration and not limitation, suitable pi selective beta blockers include Acebutolol, Atenolol, Betaxolol, Bisoprolol, Celiprolol, Metoprolol, Nebivolol, Esmolol, and Nebivolol. By means of illustration and not limitation, suitable pi selective beta blockers include Butaxamine and ICI-118,551. An exemplary 3 selective beta blocker is SR 59230A.

Thus, in certain embodiments the beta blockers is selected from the group consisting of: Propranolol, Bucindolol, Carteolol, Carvedilol, Labetalol, Nadolol, Oxprenolol, Penbutolol, Pindolol, Sotalol, Timolol, Acebutolol, Atenolol, Betaxolol, Bisoprolol, Celiprolol, Metoprolol, Nebivolol, Esmolol, Nebivolol, Butaxamine, ICI-118,551, and SR 59230A.

By means of illustration and not limitation, suitable angiotensin II receptor blockers include: Losartan and active metabolites thereof such as EXP 3174, Candesartan, Valsartan, Irbesartan, Telmisartan, Eprosartan, Olmesartan, Azilsartan, and Fimasartan.

By means of illustration and not limitation, suitable statins include: Atorvastatin, Cerivastatin, Fluvastatin, Lovastatin, Mevastatin, Pitavastatin, Pravastatin, Rosuvastatin, Simvastatin. Equally envisaged are combination therapies including statins such as but not limited to: Atorvastatin + amlodipine, Atorvastatin + perindopril + amlodipine, Lovastatin + niacin extended-release, Rosuvastatin + ezetimibe, Simvastatin + ezetimibe, Simvastatin + niacin extended-release.

The method that is used to measure the expression level of IGFBP2 and optionally the expression level of one or more additional genes disclosed herein is not limiting in the context of the present invention. For example, such methods may include biochemical assay methods, immunoassay methods, mass spectrometry analysis methods, or chromatography methods, or combinations thereof.

The method hence may comprise determining the expression level of IGFBP2 and optionally the expression level of one or more additional genes disclosed herein in the biological sample which are compared to the expression levels of the same genes in a control biological sample on the transcript level. The term “transcript” as used herein refers to a segment (i.e. sequence, stretch, concatenation) of RNA. A preferred biological sample in embodiments wherein the transcript level is measured is a serum sample.

Alternatively, the method may comprise determining the expression level of IGFBP2 and optionally the expression level of one or more additional genes disclosed herein in the biological sample which are compared to the expression levels of the same genes in a control biological sample on the protein level. A preferred biological sample in embodiments wherein the protein level is measured is a serum sample.

Yet alternatively, the method may comprise determining the expression levels of the genes in the biological sample which are compared to the expression levels of the same genes in a reference biological sample on both the transcript level and the protein level. A preferred biological sample in embodiments wherein the protein level is measured is a serum sample.

Depending on factors that can be evaluated and decided on by a skilled person, such as but not limited to the type of a marker (e.g. peptide, polypeptide, protein, or nucleic acid), the type of the biological sample, the expected abundance of the marker in the tested object, the type, robustness, sensitivity and/or specificity of the detection method used to detect the marker, the quantity and/or activity of a marker may be measured directly in the tested object, or the tested object may be subjected to one or more processing steps aimed at achieving an adequate measurement of the marker.

The term “gene” is well-known in the art and in general refers to a locatable region of genomic sequence, corresponding to a unit of inheritance, which is associated with regulatory regions, transcribed regions and/or other functional sequence regions. Genes typically comprise a coding sequences encoding a gene product, such as an RNA molecule or a polypeptide.

“Protein” as used throughout this specification generally encompasses macromolecules comprising one or more polypeptide chains, i.e. polymeric chains of amino acid residues linked by peptide bonds. The term may encompass naturally, recombinantly, semi-synthetically or synthetically produced proteins. The term also encompasses proteins that carry one or more co- or post-expression-type modifications of the polypeptide chain(s), such as, without limitation, glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-terminal methionine removal, conversion of pro-enzymes or pre-hormones into active forms, etc. The term further also includes protein variants or mutants which carry amino acid sequence variations vis-a-vis a corresponding native proteins, such as, e.g. amino acid deletions, additions and/or substitutions. The term contemplates both full-length proteins and protein parts or fragments, e.g. naturally-occurring protein parts that ensue from processing of such full-length proteins. Thus, in the context of the present invention IGFBP2 variants are equally envisaged.

The method according to the invention wherein the expression level of IGFBP2 and optionally one or more additional genes are measured may include (i.e. comprise) any commonly used technique of manipulation. Non-limiting examples hereof include RNA separation, detection and/or quantification markers at the RNA level. “RNA level” or “transcript” level as used herein may refer to the RNA as such, but may equally refer to related nucleic acid sequences. Non-limiting examples hereof include hnRNA, pre-mRNA, mRNA, or cDNA. Standard quantitative RNA or cDNA measurement tools known in the art may be used. By means of illustration and not limitation, examples thereof include hybridisation-based analysis, microarray expression analysis, digital gene expression profiling (DGE), RNA-in-situ hybridisation (RISH), Northern-blot analysis and the like, Polymerase Chain Reaction (PCR), supported oligonucleotide detection, pyrosequencing, polony cyclic sequencing by synthesis, simultaneous bi-directional sequencing, single-molecule sequencing, single molecule real time sequencing, true single molecule sequencing, hybridization-assisted nanopore sequencing, sequencing by synthesis, or single-cell RNA sequencing (scRNA seq).

The term “polymerase chain reaction”, commonly abbreviated as “PCR”, is well known to a skilled person and generally encompasses any in vitro process for increasing the number of copies of a target nucleic acid region within a nucleic acid molecule, preferably within a DNA molecule, by the action of a nucleic acid polymerase, e.g. DNA polymerase. The process may encompass both linear and exponential amplification, and particularly preferably refers to exponential amplification. In PCR, target nucleic acid region within a nucleic acid molecule, especially within a DNA molecule, is amplified using thermostable DNA polymerase(s) and at least two amplification primers, one complementary to the (+)-strand at one end of the target sequence to be amplified and the other complementary to the (-)- strand at the other end of the target sequence. When a reference to the general term “PCR” is made in the present disclosure, it is evident that this encompasses any type of PCR reaction. Thus, modifications of the prototypic PCR are envisaged. Non-limiting examples hereof include high-fidelity PCR, hot-start PCR, touch-down PCR, nested PCR, multiplex PCR, quantitative PCR, quantitative real-time PCR, long-range PCR, reverse transcription PCR RT-PCR, end-point PCR, digital PCR, digital droplet PCR, and RT-quantitative PCR (qPCR). Different PCR approaches ad methods have been described at numerous occasions in the art (e.g. in PCR Protocols: A Guide to Methods and Applications, eds. Innis et al., Academic Press, San Diego, 1990; and in Green and Sambrook, Cold Spring Hard Protoc, 2019).

The reaction can be performed in any thermocycler commonly used for PCR. However, preferred are cyclers with real-time fluorescence measurement capabilities, for example, Smartcycler® (Cepheid, Sunnyvale, CA), ABI PRISM 7700® (Applied Biosystems, Foster City, CA), Rotor-Gene TM (Corbett Research, Sydney, Australia), Lightcycler® (Roche Diagnostics Corp, Indianapolis, IN), iCycler® (Biorad Laboratories, Hercules, CA), MX4000® (Stratagene, La Jolla, CA), and CFX96 Real-Time PCR system (Biorad).

Alternatively or complementary to detection at the transcript level, the method may include techniques for separating, detecting and/or quantifying IGFBP2 and optionally one or more additional genes described herein at the protein level. Such methods are well known in the art and include without limitation immunological assay methods, wherein the ability of an assay to separate, detect and/or quantify a peptide, polypeptide, or protein is conferred by specific binding between a separable, detectable and/or quantifiable binding agent such as an immunological binding agent and the peptide, polypeptide, or protein. Immunological assay methods include without limitation immunohistochemistry, immunofluorescence, immunocytochemistry, flow cytometry, mass cytometry, fluorescence activated cell sorting (FACS), fluorescence microscopy, fluorescence based cell sorting using microfluidic systems, immunoaffinity adsorption based techniques such as the illustrative examples of affinity chromatography, magnetic particle separation, magnetic activated cell sorting or bead based cell sorting using microfluidic systems, enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immune absorbent spot (ELISPOT) based techniques, radioimmunoassay (RIA), and Western blot.

In any of the foregoing embodiments, the method described herein may include chromatography methods. The term “chromatography” encompasses any method for separating substances, such as chemical or biological substances, e.g. markers, such as preferably peptides, polypeptides, or proteins, referred to as such and vastly available in the art. In a preferred approach, chromatography refers to a process in which a mixture of substances (analytes) carried by amoving stream of liquid or gas (“mobile phase”) is separated into components as a result of differential distribution of the analytes, as they flow around or over a stationary liquid or solid phase (“stationary phase”), between said mobile phase and said stationary phase. The stationary phase may be usually a finely divided solid, a sheet of filter material, or a thin film of a liquid on the surface of a solid, or the like. Chromatography is also widely applicable for the separation of chemical compounds of biological origin, such as, e.g. amino acids, proteins, fragments of proteins or peptides, etc.

The particular about the chromatography methods that can be used are not particularly limited and may therefore be columnar (i.e. wherein the stationary phase is deposited or packed in a column), preferably liquid chromatography, and yet more preferably HPLC. Chromatography methods are well known to a person skilled in the art (see e.g. Meyer M., 1998, ISBN: 047198373X, and “Practical HPLC Methodology and Applications”, Bidlingmeyer, B. A., John Wiley & Sons Inc., 1993). Exemplary types of chromatography include, without limitation, high-performance liquid chromatography (HPLC), normal phase HPLC (NP-HPLC), reversed phase HPLC (RP-HPLC), ion exchange chromatography (IEC), such as cation or anion exchange chromatography, hydrophilic interaction chromatography (HILIC), hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC) including gel filtration chromatography or gel permeation chromatography, chromatofocusing, affinity chromatography such as immunoaffinity, and immobilised metal affinity chromatography.

Lurther techniques for separating, detecting and/or quantifying markers, such as preferably peptides, polypeptides, or proteins, may be used, optionally in conjunction with any of the above described analysis methods. Such methods include, without limitation, chemical extraction partitioning, isoelectric focusing (IEL) including capillary isoelectric focusing (CIEL), capillary isotachophoresis (CITP), capillary electrochromatography (CEC), and the like, one-dimensional polyacrylamide gel electrophoresis (PAGE), two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), capillary gel electrophoresis (CGE), capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), and free flow electrophoresis (FFE).

Optionally, the method described herein uses mass spectrometry to detect and optionally quantify the IGFBP2 expression level. The term “mass spectrometry” as used herein broadly refers to techniques that are capable of measuring mass-to-charge ratios (commonly indicated in the art by “m/z” or “m/Q”) of ions. Said techniques are well-known to a person skilled in the art. Generally, mass spectrometers comprise three main components: an ion source, a mass analyser, and a detector. In a first step ions are generated of the analyte which may optionally involve fragmentation. Subsequently, the ions are separated from each other based on mass-to-charge ratio. Finally, detection occurs by a detector. Non- limiting ionization techniques include electrospray ionization (ESI), Atmospheric Pressure Chemical Ionization, Atmospheric Pressure Photoionization, matrix-assisted laser desorption/ionization (MALDI), Gas-Phase Protonation, Ambient Desorption Ionization, Desorption Electrospray Ionization (DESI), Direct Analysis in Real Time (DART), and fast atom bombardment (FAB) (e.g. reviewed in Awad et al, Appl Spectrosc Rev, 2014). Non-limiting examples of mass selectors include Time-of- Flight (TOF) mass fdters, quadrupole mass fdters, ion trap mass filters, Fourier-transform ion cyclotron resonance mass selectors, such as orbitrap mass filters. Non-limiting examples of ion detectors include electron multipliers, Faraday cups, photomultiplier conversion dynode, and array detectors.

Examples of mass spectrometry methods suitable for use in the method subject of the invention include fast atom bombardment mass spectrometry (FAB-MS), liquid chromatography mass spectrometry (LC- MS), liquid chromatography tandem mass spectrometry (LC-MS/MS), matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS). A particularly preferred means for detecting and optionally quantifying IGFBP2 is liquid chromatography tandem mass spectrometry (LC-MS/MS). LC- MS/MS is a coupled liquid chromatography - mass spectrometry system wherein a liquid sample is analysed by mass spectrometry after separation of the sample constituents by chromatography.

Optionally, the mass spectrometry method that may be used as detection means is a targeted tandem mass spectrometry method. Targeted mass spectrometry methods are typically performed on triple quadrupole (i.e. QQQ) mass spectrometers wherein the first quadrupole (QI) acts as a filter to select predicted precursor molecules, the second quadrupole (Q2) is used as a collision cell to fragment said precursor (i.e. parent) molecules, and the third quadrupole (Q3) detects a predefined fragment m/z (i.e. the daughter molecules). Alternatively targeted tandem mass spectrometry approaches have been described in the art and include quadrupole-orbitrap approaches (Vidova and Spacil, Anal Chim Acta, 2017). The precise targeted tandem mass spectrometry method is not particularly limiting and may therefore be selected from reaction monitoring (SRM), multiple reaction monitoring (MRM), or parallel reaction monitoring (PRM).

A skilled person appreciates that in several of the above-mentioned methods and techniques that allow for quantitation of the IGFBP2 expression level and optionally one or more additional genes described herein, at least one step is present which resides in specific binding of one or more binding agents to said genes. Thus, in such embodiments the method comprises the use of one or more binding agents (i.e. a molecule capable of specifically binding to one of the genes or its gene product). Such binding agents may be in various forms, including a lyophilized form, free in solution, or immobilized form on for example a solid phase such as beads or an arrays. They may be, e.g. provided in a multi -well plate or as an array or microarray, or they may be packaged separately, individually, or in combination. The term “specifically bind” as used throughout this specification means that an agent (denoted herein also as “binding agent” or “specific-binding agent”) binds to one or more desired molecules or analytes (e.g. peptides, polypeptides, proteins, or nucleic acids) substantially to the exclusion of other molecules which are random or unrelated, and optionally substantially to the exclusion of other molecules that are structurally related. The term “specifically bind” does not necessarily require that an agent binds exclusively to its intended target(s). For example, an agent may be said to specifically bind to target(s) of interest if its affinity for such intended target(s) under the conditions of binding is at least about 2- fold greater, preferably at least about 5 -fold greater, more preferably at least about 10-fold greater, yet more preferably at least about 25-fold greater, still more preferably at least about 50-fold greater, and even more preferably at least about 100-fold, or at least about 1000-fold, or at least about 10⁴-fold, or at least about 10⁵-fold, or at least about 10⁶-fold or more greater, than its affinity for a non-target molecule, such as for a suitable control molecule (e.g. bovine serum albumin, casein).

Suitable “binding agents” as intended throughout this specification suitable for determining IGFBP2 expression levels include by means of illustration antibodies, antibody fragments, antibody-like protein scaffolds, aptamers, spiegelmers (L-aptamers), photo aptamers, proteins, peptides, peptidomimetics, nucleic acids such as oligonucleotides (for example hybridization probes, amplification primers, sequencing primers, and primer pairs), small molecules, and any combination thereof.

Preferably, the specific binding agent may bind to its intended target(s) with affinity constant (KA) of such binding KA > IxlO⁶ M ¹, more preferably KA > IxlO⁷ M ¹, yet more preferably KA > IxlO⁸ M ¹, even more preferably KA > IxlO⁹ M ¹, and still more preferably KA > IxlO¹⁰ M ¹ or KA > IxlO¹¹ M ¹ or KA > IxlO¹² M ¹, wherein KA = [SBA_T]/[SBA][T], SBA denotes the specific-binding agent, and T denotes the intended target. Determination of KA can be carried out by methods known in the art, such as for example, using equilibrium dialysis and Scatchard plot analysis.

As used herein, the term “antibody” is used in its broadest sense according to its common interpretation in the art and generally refers to any immunologic binding agent. The term specifically encompasses intact monoclonal antibodies, polyclonal antibodies, multivalent (such as but not limited to 2-, 3- or more-valent) and/or multi-specific antibodies (i.e. bi- or more-specific antibodies) formed from at least two intact antibodies, and antibody fragments insofar they exhibit the desired biological activity (particularly, ability to specifically bind an antigen of interest, i.e. antigen-binding fragments), as well as multivalent and/or multi-specific composites of such fragments. The term “antibody” is not only inclusive of antibodies generated by methods comprising immunisation, but also includes any polypeptide, e.g. a recombinantly expressed polypeptide, which is made to encompass at least one complementarity-determining region (CDR) capable of specifically binding to an epitope on an antigen of interest. An antibody may be any of IgA, IgD, IgE, IgG and IgM classes, and preferably IgG class antibody. The term antibody includes antibodies originating from or comprising one or more portions derived from any animal species, preferably vertebrate species, including, e.g. birds and mammals. Without limitation, the antibodies may be chicken, turkey, goose, duck, guinea fowl, quail or pheasant. Also without limitation, the antibodies may be human, murine (e.g. mouse, rat, etc.), donkey, rabbit, goat, sheep, guinea pig, camel (e.g. Camelus bactrianus and Camelus dromaderius), llama (e.g. Lama paccos, Lama glama or Lama vicugna) or horse. An antibody may be a polyclonal antibody, such as an antiserum or immunoglobulins purified from antiserum. An antibody may be a monoclonal antibody or a mixture of monoclonal antibodies. Monoclonal antibodies can target a particular antigen or a particular epitope within an antigen with greater selectivity and reproducibility.

The term “immunoglobulin” or “immunoglobulin sequence” is used as a general term to include both the full-size antibody, the individual chains thereof, as well as all parts, domains or fragments thereof (including but not limited to antigen-binding domains or fragments such as VHH domains or VH/VL domains, respectively). In addition, the term “sequence” as used herein (for example in terms like “immunoglobulin sequence”, “antibody sequence”, “variable domain sequence”, “VHH sequence” or “protein sequence”), should generally be understood to include both the relevant amino acid sequence as well as nucleic acid sequences or nucleotide sequences encoding the same, unless the context requires a more limited interpretation.

A full-length antibody as existing naturally is an immunoglobulin molecule comprising 2 heavy (H) chains and 2 light (L) chains interconnected by disulfide bonds. The amino terminal portion of each chain includes a variable region of about 100-110 amino acids primarily responsible for antigen recognition via the complementarity determining regions (CDRs) contained therein. The carboxyterminal portion of each chain defines a constant region primarily responsible for effector function.

The term “polyclonal antibody” as used herein may be an antiserum or immunoglobulins purified there from (e.g. affinity-purified). Conversely, the term “monoclonal antibody” refers to an antibody that is derived from a single copy or clone including, for example, any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Monoclonal antibodies can target a particular antigen or a particular epitope within an antigen with greater selectivity and reproducibility. Monoclonal antibodies preferably exist in a homogeneous or substantially homogeneous population. Monoclonal antibodies and antigen-binding fragments thereof of the present invention can be produced, for example, by recombinant technologies, phage display technologies, synthetic technologies (e.g. CDR-grafting), or combinations of such technologies, or other technologies known in the art.

Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art, as are methods to produce recombinant antibodies or fragments thereof (e.g. Harlow and Lane, “Antibodies: A Laboratory Manual”, Cold Spring Harbour Laboratory, New York, 1988). By means of illustration and not limitation, monoclonal antibodies may be made by the hybridoma method first described by Kohler et al. (Nature, 1975), or may be made by recombinant DNA methods (detailed inter alia in US 4,816,567). Monoclonal antibodies may also be made using phage antibody libraries using techniques as described by Clackson et al. (Nature, 1991) and Marks et al. (J Mol Biol, 1991).

The term antibody includes antibodies originating from or comprising one or more portions derived from any animal species, preferably vertebrate species, including, e.g. birds and mammals. Without limitation, the antibodies may be chicken, turkey, goose, duck, guinea fowl, quail or pheasant. Also without limitation, the antibodies may be human, murine (e.g. mouse, rat, etc.), donkey, rabbit, goat, sheep, guinea pig, camel (e.g. Camelus bactrianus and Camelus dromaderius), llama (e.g. Lama paccos, Lama glama or Lama vicugna) or horse.

The term antibody as used herein also encompasses “chimeric antibodies” which originate from at least two animal species. The term “chimeric antibody” or “chimeric antibodies” refers to antibodies which comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as for example antibodies having murine heavy and light chain variable regions linked to human, canine, equine, or feline constant regions. Chimeric antibodies comprise a portion of the heavy and/or light chain that is identical to or homologous with corresponding sequences from antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous with corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, exhibiting the desired biological activity (e.g. Morrison et al., Proc. Natl. Acad. Sci, 1984). Chimeric antibodies are made through merging DNA encoding a portion, such as the Fv region, of a monoclonal antibody from one species, e.g. mouse or monkey, with the antibodyproducing DNA from another species, e.g. human.

The term antibody as used herein also encompasses “fully human antibodies”. The term “human antibody” or “fully human antibody” refers to an antibody of which the encoding genetic information is of human origin. Accordingly, the term “fully human antibody” refers to antibodies having variable and constant regions derived only from human germline immunoglobulin sequences. The term “fully human antibody” is thus not to include antibodies in which CDR sequences derived from the germline of other mammalian species, such as a mouse, have been grafted onto human framework sequences.

The term antibody as used herein also encompasses “humanized antibodies”. The term “humanized antibody” refers to antibodies derived from non-human species whose protein sequence have been modified so as to increase their similarity to antibodies produced naturally in humans, more particularly, antibodies which comprise heavy and light chain variable region sequences from a non -human species (e.g. a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “human-like”, i.e. more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which non-human CDR sequences are introduced into human VH and VL sequences to replace the corresponding human CDR sequences.

The humanized antibody is an antibody or a variant, derivative, analogue or fragment thereof which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-human antibody. A humanized antibody comprises substantially all, or at least one, and typically two, variable domains (Fab, Fab', F(ab') 2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non- human immunoglobulin (i.e. donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. A humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. A humanized antibody may contain both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CHI , hinge, CH2, CH3, and CH4 regions of the heavy chain. Alternatively, a humanized antibody may only contain a humanized light chain, or a humanized heavy chain. An exemplary humanized antibody contains a humanized variable domain of a light chain and a humanized variable domain of a heavy chain.

A skilled person will understand that an antibody can include one or more amino acid deletions, additions and/or substitutions (e.g. conservative substitutions), insofar such alterations preserve its binding of the respective antigen. For example, mutations may be introduced into the antibody, in particular in the Fc region, to extend in vivo half-life without compromising immunogenicity as described in US patent 8,323,962. An antibody may also include one or more native or artificial modifications of its constituent amino acid residues (e.g. glycosylation, etc.).

Antibody binding agents may be antibody fragments. “Antibody fragments” comprise a portion of an intact antibody, comprising the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab’, F(ab’)2, Fv and scFv fragments, single domain (sd) Fv, such as VH domains, VL domains and VHH domains; diabodies; linear antibodies; single-chain antibody molecules, in particular heavy-chain antibodies; and multivalent and/or multispecific antibodies formed from antibody fragment(s), e.g. dibodies, tribodies, and multibodies. The above designations Fab, Fab’, F(ab’)2, Fv, scFv etc. are intended to have their art-established meaning.

The term “antibody-like protein scaffolds” or “engineered protein scaffolds” broadly encompasses proteinaceous non-immunoglobulin specific-binding agents, typically obtained by combinatorial engineering (such as site-directed random mutagenesis in combination with phage display or other molecular selection techniques). Usually, such scaffolds are derived from robust and small soluble monomeric proteins (such as Kunitz inhibitors or lipocalins) or from a stably folded extra-membrane domain of a cell surface receptor (such as protein A, fibronectin or the ankyrin repeat).

The term “aptamer” refers to single-stranded or double-stranded oligo-DNA, oligo-RNA or oligo- DNA/RNA or any analogue thereof that specifically binds to a target molecule such as a peptide. Advantageously, aptamers display fairly high specificity and affinity (e.g. KA in the order IxlO⁹ M ¹) for their targets. Aptamer production has been is described in detail in the art (e.g. in Ellington & Szostak, Nature 1990; and Tuerk & Gold, Science, 1990). The term “photo aptamer” refers to an aptamer that contains one or more photoreactive functional groups that can covalently bind to or crosslink with a target molecule. The related term “spiegelmer” refers to an aptamer which includes L- DNA, L-RNA, or other left-handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides. The term “peptidomimetic” refers to a non-peptide agent that is a topological analogue of a corresponding peptide. Methods of rationally designing peptidomimetics of peptides have been described in the art (e.g. Horwell, Trends Biotechnol, 1995).

As described above, the term “oligonucleotide” as used throughout this specification refers to a nucleic acid (including nucleic acid analogues and mimetics) oligomer or polymer (i .e . a nucleic acid sequence) . In the context of binding agents, the oligonucleotide is preferably an antisense oligonucleotide which is single-stranded or substantially single stranded. Oligonucleotides as intended herein may be preferably between about 10 and about 100 nucleoside units (i.e. nucleotides or nucleotide analogues) in length, preferably between about 15 and about 50, more preferably between about 20 and about 40, also preferably between about 20 and about 30. Oligonucleotides as intended herein may comprise one or more or all non-naturally occurring heterocyclic bases and/or one or more or all non-naturally occurring sugar groups and/or one or more or all non-naturally occurring inter-nucleoside linkages, the inclusion of which may improve properties such as, for example, increased stability in the presence of nucleases and increased hybridization affinity, increased tolerance for mismatches, etc.

Nucleic acid binding agents, such as oligonucleotide binding agents, are typically at least partly antisense to a target nucleic acid of interest. The term “antisense” generally refers to an agent (e.g. an oligonucleotide) configured to specifically anneal with (hybridize to) a given sequence in a target nucleic acid, such as for example in a target DNA, hnRNA, pre-mRNA or mRNA, and typically comprises, consists essentially of or consists of a nucleic acid sequence that is complementary or substantially complementary to said target nucleic acid sequence. Antisense agents suitable for use herein, such as hybridization probes or amplification or sequencing primers and primer pairs) may typically be capable of annealing with (hybridizing to) the respective target nucleic acid sequences at high stringency conditions, and capable of hybridizing specifically to the target under physiological conditions. The terms “complementary” or “complementarity” as used throughout this specification with reference to nucleic acids, refer to the normal binding of single-stranded nucleic acids under permissive salt (ionic strength) and temperature conditions by base pairing, preferably Watson-Crick base pairing. By means of example, complementary Watson-Crick base pairing occurs between the bases A and T, A and U or G and C. For example, the sequence 5'-A-G-U-3' is complementary to sequence 5'-A-C-U-3'.

The reference to oligonucleotides may in particular but without limitation include specifically hybridisable probes and/or amplification primers and/or sequencing primers, etc., as commonly used in nucleic acid detection technologies. “Specifically hybridisable” and “specifically complementary” are terms that indicate a sufficient degree of complementarity such that stable and specific binding occurs between the oligonucleotide (or its analogue) and the DNA, RNA, and or DNA-RNA hybrid target. The oligonucleotide or oligonucleotide analogue need not be 100% complementary to its target sequence to be specifically hybridisable. An oligonucleotide or analogue is specifically hybridisable when there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide or analogue to non-target sequences under conditions where specific binding is desired. Such binding is referred to as specific hybridization.

Probes may be preferably be less than or equal to about 50 nucleotides in length, for example less than or equal to about 40, about 30, about 20, or less than about 10 nucleotides in length, e.g. between 10 and 30 or between 15 and 25 nucleotides in length.

A probe comprises an oligonucleotide sequence which effects the hybridisation (annealing) of the probe with a sequence comprised in a nucleic acid to be detected by the probe. In certain embodiments, a probe does not contain any further oligonucleotide sequence(s). In certain other embodiments, a probe may contain - besides the oligonucleotide sequence which effects the hybridisation of the probe with a sequence comprised in a nucleic acid to be detected by the probe - additional oligonucleotide sequence(s) serving other useful purpose(s). For example but without limitation, such additional oligonucleotide sequence(s) may provide linker sequences allowing to couple a probe with another moiety or moieties, e.g. label(s) or reporter moiety, e.g. a radioactive isotope (e.g. 32P, 33P), ligand, chemiluminescent agent, fluorophore (e.g. fluorescein, tetrachloro-fluorescein, TAMRA, ROX, Cy3, Cy3.5, Cy5, Cy5.5, Texas Red, etc.), vitamin (e.g. biotin), steroid (e.g. digoxin), enzyme (e.g. HRP, AP, etc.), etc., or may provide sequences ensuring a certain conformation of a probe, etc.; various options are available to a skilled reader. By means of example and not limitation, when a probe forms a molecular beacon as known in the art, mutually complementary oligonucleotide extensions are provided at the 5’ and 3’ ends of the probe, one of the oligonucleotide extensions linked to a fluorophore (e.g. fluorescein, carboxyfluorescein (FAM), tetrachloro-fluorescein, TAMRA, ROX, Cy3, Cy3.5, Cy5, Cy5.5, Texas Red, etc.) and the other one to a quencher (e.g. ZENTM internal quencher, 3’ Iowa Black Black® FQ quencher) capable of quenching the fluorescent emission of the fluorophore. When the probe is not annealed to the nucleic acid to be detected, the mutually complementary oligonucleotide extensions will form a hairpin structure, whereby the quencher is brought into proximity of the fluorophore and quenches the fluorophore’s signal. Conversely, when the probe is annealed to the nucleic acid to be detected, the hairpin structure cannot formed, the quencher is not in proximity of the fluorophore and does not quench the fluorophore’s signal, which signal is therefore detectable.

The term “small molecule” refers to compounds, preferably organic compounds, with a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g. proteins, propeptides, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to about 4000, more preferably up to 3000 Da, more preferably up to 2000 Da, even more preferably up to about 1000 Da, most preferably up to about 500 Da.

Any of the binding agents to IGFBP2 or one of the optional further genes described herein may comprise a detectable label. The term “label” refers to any atom, molecule, moiety or biomolecule that may be used to provide a detectable and preferably quantifiable read-out or property, and that may be attached to or made part of an entity of interest, such as a binding agent. Labels may be suitably detectable by for example mass spectrometric, spectroscopic, optical, colorimetric, magnetic, photochemical, biochemical, immunochemical or chemical means. Labels include without limitation dyes; radiolabels such as ³²P, ³³P, ³⁵S, ¹²⁵1, ¹³¹I; electron-dense reagents; enzymes (e.g. horse-radish peroxidase or alkaline phosphatase as commonly used in immunoassays); binding moieties such as biotin-streptavidin; haptens such as digoxigenin; luminogenic, phosphorescent or Anorogenic moieties; mass tags; and fluorescent dyes alone or in combination with moieties that may suppress or shift emission spectra by fluorescence resonance energy transfer (FRET).

In some embodiments, binding agents may be provided with a tag that permits detection with another agent (e.g. with a probe binding partner). Such tags may be, for example, biotin, streptavidin, his-tag, myc tag, maltose, maltose binding protein or any other kind of tag known in the art that has a binding partner. Non-limiting example of associations which may be utilised in the probe:binding partner arrangement may be any, and includes, for example biotin: streptavidin, his-tag:metal ion (e.g. Ni²⁺), and maltose maltose binding protein. The marker-binding agent conjugate may be associated with or attached to a detection agent to facilitate detection. Examples of detection agents include, but are not limited to, luminescent labels; colorimetric labels, such as dyes; fluorescent labels; or chemical labels, such as electroactive agents (e.g. ferrocyanide); enzymes; radioactive labels; or radiofrequency labels. The detection agent may be a particle. Examples of such particles include, but are not limited to, colloidal gold particles; colloidal sulphur particles; colloidal selenium particles; colloidal barium sulphate particles; colloidal iron sulphate particles; metal iodate particles; silver halide particles; silica particles; colloidal metal (hydrous) oxide particles; colloidal metal sulphide particles; colloidal lead selenide particles; colloidal cadmium selenide particles; colloidal metal phosphate particles; colloidal metal ferrite particles; any of the above-mentioned colloidal particles coated with organic or inorganic layers; protein or peptide molecules; liposomes; or organic polymer latex particles, such as polystyrene latex beads.

RT-PCR is a suitable method to determine RNA expression levels (such as mRNA expression levels) of the genes described herein. Detailed protocols and considerations relating to RT-PCR have been described at numerous instances in the art (e.g. by VanGuilder et al., Biotechniques, 2018). In brief, the main steps of an RT-PCR in the context of the present disclosure comprise: a first step of RNA extraction and/or isolation, a subsequent step of reverse transcription of the mRNA to a complementary DNA (cDNA), and an exponential amplification step of the cDNA prior to detection. Means and methods for mRNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology (for example Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons, 1997). When the RNA is or comprises mRNA, miRNA or other types of RNA, RT-PCR typically includes reverse transcription of the RNA template into cDNA, followed by amplification of the cDNA in a “traditional” PCR reaction by means of a DNA polymerase. Commonly used reverse transcriptases include, but are not limited to, avilo myeloblastosis virus reverse transcriptase (AMV- RT) and Moloney murine leukaemia virus reverse transcriptase (MMLV- RT). The reverse transcription step may rely on the use of specific primers, random hexamers, oligo-dT primers, or any combination thereof. To arrive at a typical PCR amplicon, two oligonucleotide primers (i.e. a primer pair) are generally used, as a skilled person appreciates.

The term “primer pair” or “amplification primer pair” refers to a combination of two primers which are suited for amplification of a target nucleic acid region (amplicon) from within a nucleic acid of interest by a polymerase-based amplification process, e.g. PCR. The ability to amplify an amplicon from within the nucleic acid of interest using a primer pair designed to specifically hybridise within the nucleic acid indicates the presence (and optionally quantity) of the nucleic acid in the polymerase-based amplification reaction.

Real time quantitative PCR (also quantitative real time polymerase chain reaction, QRT-PCR or Q- PCR) is a more recent variation of the RT-PCR technique. Q-PCR is capable of measuring PCR product accumulation through a dual -labelled fluorogenic probe. Thus, in embodiments of the method described herein wherein a quantitative PCR reaction is conducted, a third oligonucleotide (i.e. probe) is designed to detect a nucleotide sequence located between the two PCR primers. Said probe is generally nonextendible by DNA polymerase enzyme, and may be labelled with a reporter fluorescent dye and a quencher fluorescent dye (cf. a Taqman assay). In these embodiments, any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together as they are on the probe. During the amplification reaction, the DNA polymerase enzyme (in Taqman assays routinely a Taq polymerase which is characterised by a 5 '-3' nuclease activity but does not comprise a 3 '-5' proofreading endonuclease activity) cleaves the probe in a template-dependent manner. The resultant probe fragments disassociate in solution, and a signal from the released reporter dye is effectively rendered free from the quenching effect of the second fluorophore. One molecule of reporter dye is released for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data.

An alternative preferred means for detecting and comparing expression levels of one or more genes selected from the panel of genes is by means of one or more microarrays, such as an oligonucleotide array or a protein array. The use of microarrays has been described extensively in the art at numerous occasions and is therefore known to a skilled person (e.g. Slonim and Yanai, PLoS Comput Biol, 2009). Arrays typically contain addressable moieties that can detect the presence (or absence) of one or more entities (in the context of the present invention transcripts or protein gene products) in one or more samples, e.g. via a binding event. Microarrays include without limitation DNA microarrays, such as cDNA microarrays, oligonucleotide microarrays, SNP microarrays, microRNA arrays, protein microarrays, antibody microarrays, tissue microarrays, cellular microarrays (also called transfection microarrays), chemical compound microarrays, and carbohydrate arrays (glycoarrays).

DNA arrays comprise a collection of (optionally customisable) nucleotide sequences that can bind to (target) sequences present in a sample. While microarrays in general each rely on this principle, distinct array assays can be developed for different purposes. Alternatively, protein microarrays are suitable to identify protein-protein interactions, including without limitation identifying substrates of protein kinases, transcription factor protein- activation, or to identify the targets of biologically active small molecules. Protein arrays may comprise an array of different protein molecules, commonly antibodies, or nucleotide sequences that bind to proteins of interest. Antibody microarrays comprise antibodies spotted onto the protein chip that are used as capture molecules to detect proteins or other biological materials from a sample, e.g. from cell or tissue lysate solutions. For example, antibody arrays can be used to detect biomarkers from bodily fluids, e.g. serum or urine, for diagnostic applications. Tissue microarrays comprise separate tissue cores assembled in array fashion to allow multiplex histological analysis. Cellular microarrays, also called transfection microarrays, comprise various capture agents, such as antibodies, proteins, or lipids, which can interact with cells to facilitate their capture on addressable locations. Chemical compound microarrays comprise arrays of chemical compounds and can be used to detect protein or other biological materials that bind the compounds. Carbohydrate arrays (glycoarrays) comprise arrays of carbohydrates and can detect, e.g. protein that bind sugar moieties. A skilled person will appreciate that each of the above arrays is suitable for a subset of embodiments defined herein and therefore appreciates their use according to and adapted for the methods of the invention.

A biomarker microarray panel can be processed in manual, semi-automatic or automatic modes. Manual mode refers to manual operations for all assay steps including reagent and sample delivery onto microarrays, sample incubation and microarray washing. Semi-automatic modes refer to manual operation for sample and reagent delivery onto microarray, while incubation and washing steps operate automatically. In an automatic mode, three steps (sample/reagent delivery, incubation and washing) can be controlled by a computer or similar apparatus.

Related to the above, also envisaged is the assessment of functional gene expression levels of IGFBP2 and optionally one of the additional genes described herein in a biological sample of a subject. “Functional” in this context refers to the capacity of the gene, or its gene product to exert the normal function of that gene or its gene product. Subsequently, the term “biological function” or “function” as used herein is to be interpreted broadly and may generally encompass any one or more aspects of the biological function of the target at any level (e.g. molecular, cellular and/or physiological), such as without limitation any one or more aspects of its biochemical activity, signalling activity, interaction activity, receptor activity or structural activity (e.g. in or on a cell, cell population, tissue, organ, or organism, e.g. in a biological sample from a subject). Hence, while the expression levels of IGFBP2 and optionally one of the additional genes described herein are indicative for the absolute amount of expression, the term may also encompass the expression level of a properly functioning gene or its gene product.

In certain embodiments of the method, the method includes measuring the expression levels of one or more isoforms of IGFBP2. A skilled person readily appreciates that depending on retainment of their functionality, isoforms are usually suitable for inclusion as part of the overall expression level of IGFBP2. Thus, in certain further embodiments the one or more isoforms are considered to add to the expression level of IGFBP2. In certain alternative embodiments, the one or more isoforms are not considered to add to the expression level of IGFBP2. In certain further embodiments the one or more isoforms are considered to add to the expression level of the IGFBP2. In certain alternative embodiments, the one or more isoforms are not considered to add to the expression level of IGFBP2. A yet alternative suitable technique to detect IGFBP2 expression and optionally one or more of the additional genes described herein is by means of immunohistochemistry. “Immunohistochemistry” refers to a process of localising IGFBP2 in cells of a tissue (such as a biopsy, or a microscopic coupe of a biopsy) by binding antibodies specifically to IGFBP2 that may be expressed by or in the tissues. The antigen-binding antibody can be conjugated or fused to any suitable tag such as described above that allows its detection, (e.g. by visualization). The tag may be an enzyme that can catalyse a colourproducing reaction, such as alkaline phosphatase or horseradish peroxidase. The enzyme can be fused to the antibody or non-covalently bound, e.g. using a biotin-avidin system. Alternatively, the antibody can be tagged with a fluorophore, such as fluorescein, rhodamine, DyLight Fluor or Alexa Fluor. The antigen-binding antibody can be directly tagged or it can itself be recognized by a detection antibody that carries the tag. Immunohistochemistry detection may be multiplexed to allow simultaneous or near simultaneous detection of multiple proteins in a single sample. In certain embodiments, the immunohistochemistry method is conducted such that the expression level of a gene product such as IGFBP2 is related to its staining intensity.

As is evident for any skilled person based on the present disclosure, the method of the invention described herein may incorporate or rely on use of computer-assisted detection and/or analysis means such as software or computer-controlled sensors. The present invention therefore further relates to a computer system comprising a processor, and optionally a memory coupled to said processor and encoding one or more software programs, wherein said one or more software programs instruct the processor to carry out the method subject of the present disclosure. Thus, in any of the embodiments described herein, the method may be a computer-implemented method. In embodiments where the method is a computer-implemented method, the method includes obtaining by a computing device the IGFBP2 expression level in a biological sample such as a serum sample, and optionally storing, by the computing device, the probabilistic assessment (i.e. the prediction) of a thoracic aortic aneurysm based on the measured IGFBP2 expression level. The computing device may obtain the plurality of measured biomarker levels in an automated manner (i.e. without any user intervention), in a semi-automatic manner (e.g. batch input of a group of biomarker levels), or by manual user input of (each of) the measured biomarker levels.

In such embodiments, the computer software typically includes a computer readable medium having computer-executable instructions for performing the logic steps of the method of the invention. Nonlimiting examples of a suitable computer readable medium include floppy disks, CD-ROM/DVD/DVD- ROM, a hard-disk drives, flash memory, ROM/RAM, and magnetic tapes. The computer executable instructions may be written in any suitable computer language or combination of several languages. Basic computational biology methods have been described in the art and are therefore known to a skilled person (e.g. Gauthier et al., Brief Bioinform, 2019). Upon determination of the IGFBP2 expression level and optionally one or more of the additional genes in the biological sample, skilled practitioners (e.g. physicians, genetic counsellors, researcher) or the subject may be informed of the result. Optionally, the result can be cast in a transmittable form that can be communicated or transmitted to other researchers or physicians or genetic counsellors or patients. Such a form can vary and can be tangible (e.g. papers, computer readable media such as floppy disks, compact disks) or intangible (e.g. by means of email, website, or intranet). The result with regard to IGFBP2 detection, and optionally the quantitative amount thereof in the biological sample tested can be communicated for example by descriptive statements, diagrams, photographs, charts, images or any other visual forms.

Optionally, the computer implemented method provides means for generating an outcome value which is submitted in an online tool such as, but not limited to, a website or a mobile application.

The method is characterized by high sensitivity and/or specificity for the cited applications, such as detection of thoracic aortic aneurysm and/or thoracic aortic dissection. In certain embodiments, the method has a sensitivity and/or specificity (preferably sensitivity and specificity) of at least about 50%, preferably at least about 60%, preferably at least about 70%, preferably at least about 80%, more preferably at least about 85%, more preferably at least about 90%, most preferably at least about 95% to detect thoracic aortic aneurysm and/or thoracic aortic dissection.

In a further aspect the use of insulin-like growth factor binding protein 2 (IGFBP2) is envisaged for determining a thoracic aortic aneurysm, the progression of thoracic aortic aneurysm development, or for determining thoracic aortic dissection in a subject. Hence, envisaged is IGFBP2 for use in detection of thoracic aortic aneurysm, for use in monitoring the progression of thoracic aortic aneurysm development, or for use in determining thoracic aortic dissection in a subject.

In a related aspect, the invention provides a method for detecting and optionally quantifying the IGFBP2 expression level, said method comprising the steps of (i) obtaining a biological sample, preferably a serum sample from a subject; and (ii) detecting or measuring the quantity or expression level of IGFBP2 in the biological serum sample; wherein said subject has a thoracic aortic aneurysm.

In a related aspect, the invention provides a method for diagnosing and optionally treating a thoracic aortic aneurysm and/or thoracic aortic dissection in a subject comprising the steps of (i) measuring the IGFBP2 expression level in a biological sample such as a serum sample from the subject; (ii) determining whether the subject suffers from aortic aneurysm and/or thoracic aortic dissection based on said detected IGFBP2 expression level and (iii) treating said aortic aneurysm and/or thoracic aortic dissection by appropriate treatment for said aortic aneurysm and/or thoracic aortic dissection. In particular embodiments, said treatment is performing endovascular surgery or open-chest surgery. Accordingly, in particular embodiments, the method is a method of determining whether a patient is in need of treatment, such as treatment selected from the group of treatments comprising treatment with beta-blockers, sartans, endovascular surgery, and/or open-chest surgery.

In a related aspect, the invention provides a method for diagnosing and optionally treating a thoracic aortic aneurysm in a subject comprising the steps of (i) measuring the IGFBP2 expression level in a biological sample such as a serum sample from the subject; (ii) diagnosing the subject as in need of treatment of thoracic aortic aneurysm when said IGFBP2 is detected at or above a threshold value in the biological sample; and (iii) administering an effective amount of a medicament described in the art to the diagnosed subject.

In a related aspect, the invention provides a method for diagnosing and treating a thoracic aortic aneurysm in a subject comprising the steps of (i) measuring the IGFBP2 expression level in a biological sample such as a serum sample from the subject; (ii) diagnosing the subject as in need of treatment of thoracic aortic aneurysm when said IGFBP2 is detected at or above a threshold value in the biological sample; and (iii) monitoring the effect of an amount of a medicament to the diagnosed subject.

In yet an alternative aspect, the prevention provides a method for diagnosing and treating a thoracic aortic aneurysm in a subject comprising the steps of (i) determining the quantity or expression level of IGFBP2 in a biological sample such as a serum sample from the subject; (ii) comparing the quantity or expression level of IGFBP2 as determined in (i) with a reference value, said reference value representing a known diagnosis of thoracic aortic aneurysm; (iii) diagnosing the subject as in need of treatment of the thoracic aortic aneurysm when said quantity or expression level of IGFBP2 as determined in (i) deviates from said reference value; and (iv) administering an effective amount of a pharmaceutically active ingredient to the diagnosed subject.

In each of the above aspects and embodiments, the methods may involve comparing subsequent samples from the same patient in order to determine progression of thoracic aortic aneurysm or progression of the risk of developing thoracic aortic aneurysm.

The term “effective amount” as used herein may refer to a prophylactically effective amount, which is an amount of an active compound or pharmaceutical agent, more particularly a prophylactic agent, that inhibits or delays in a subject the onset of a disorder as being sought by a researcher, veterinarian, medical doctor or other clinician, or may refer to a therapeutically effective amount, which is an amount of active compound or pharmaceutical agent, more particularly a therapeutic agent, that elicits the biological or medicinal response in a subject that is being sought by a researcher, veterinarian, medical doctor or other clinician. The term “administration” or “administering” as used herein refers to the giving of a certain treatment of a thoracic aortic aneurysm to a subject in need of such a treatment. Such a treatment can be a therapeutic or prophylactic agent. The route of administration may be essentially any route of administration, such as without limitation, oral administration (e.g. oral ingestion or inhalation), intranasal administration (e.g. intranasal inhalation or intranasal mucosal application), parenteral administration (e.g. subcutaneous, intravenous, intramuscular, intraperitoneal or intrastemal injection or infusion), transdermal or transmucosal (e.g. oral, sublingual, intranasal) administration, topical administration, rectal, vaginal or intra-tracheal instillation, and the like.

In a further aspect the present invention provides a kit of parts for predicting thoracic aortic aneurysm and/or thoracic aortic dissection development. Equally envisaged is a kit of parts for diagnosing a subject with thoracic aortic aneurysm or thoracic aortic dissection. Also envisaged are kit of parts for monitoring disease progression of a thoracic aortic aneurysm and/or thoracic aortic dissection. Further envisaged are kit of parts for monitoring treatment efficacy of a medicament directed against thoracic aortic aneurysm.

The kit of parts comprises means for measuring the expression level of IGFBP2 in a biological sample such as a serum sample. Optionally, the kit of parts comprises means for measuring one or more of the optionally additional genes described in the present disclosure. In certain embodiments, the kit of parts comprises means for measuring the expression level of IGFBP2 transcript in a biological sample such as a serum sample. In certain embodiments, the kit of parts comprises means for measuring the protein expression level of IGFBP2 in a biological sample such as a serum sample. In certain embodiments, the kit of parts comprises means for measuring the expression level of one or more isoforms of IGFBP2 in a biological sample such as a serum sample.

The terms “kit of parts” and “kit” as used herein refer to a product containing components necessary for carrying out the methods (e.g. the method for detecting a thoracic aortic aneurysm and/or thoracic aortic dissection), packed so as to allow their transport and storage. Materials suitable for packing the components comprised in a kit include crystal, plastic (e.g. polyethylene, polypropylene, polycarbonate), bottles, flasks, vials, ampules, paper, envelopes, or other types of containers, carriers or supports. Where a kit comprises a plurality of components, at least a subset of the components (e.g. two or more of the plurality of components) or all of the components may be physically separated, e.g. comprised in or on separate containers, carriers or supports.

The components comprised in a kit may be sufficient or may not be sufficient for carrying out the specified method, such that external reagents or substances may not be necessary or may be necessary for performing the methods, respectively. Typically, kits are employed in conjunction with standard laboratory equipment, such as liquid handling equipment, environment (e.g. temperature) controlling equipment, analytical instruments, etc. In addition to the recited set of components as taught herein (i.e. a at least DMSO and methanol), the present kits may also include some or all of solvents, buffers. Examples of solvents of buffers include without limitation histidine-buffers, citrate-buffers, succinate- buffers, acetate-buffers, phosphate-buffers, formate buffers, benzoate buffers, TRIS (Tris(hydroxymethyl)-aminomethan) buffers or maleate buffers, or mixtures thereof. Additionally or alternatively, the kit of parts may include enzymes, detectable labels, detection reagents, and control formulations (positive and/or negative), useful in the method subject of the invention. The terms may be used interchangeably with the term “article of manufacture”, which broadly encompasses any manmade tangible structural product, when used in the present context. Typically, the kits may also include instructions for use thereof, such as on a printed insert or on a computer readable medium. The kit may further comprise documents regarding safety, documents concerning quality assurance and any other information that is commonly provided in kit of parts.

The kit of parts may comprise the means for determining the IGFBP2 expression level in a biological sample such as a serum sample in a single container or separate containers that are to be mixed by the user prior to performing the method described herein. The kit or parts may comprise said means a multiple amount of times. Optionally, the kit of parts may comprise means for preparation of a serum sample from a blood samples, and/or means for obtaining a blood sample from a subject.

The kit of parts may comprise one or more suitable control samples. By means of illustration and not limitation, the kit may therefore comprise as negative control a sample ready or substantially ready for the detection step of the method wherein said sample does not contain IGFBP2. By means of illustration and not limitation, the kit may comprise as positive control a sample ready or substantially ready for the detection step of the method wherein said sample IGFBP2 in an amount that corresponds to an amount sufficient for the method to indicate that a subject has a thoracic aortic aneurysm, or is at risk of developing a thoracic aortic aneurysm. In embodiments where in addition to IGFBP2 further expression levels of the one or more optional biomarkers are detected and/or quantified, the kit of parts may comprise a single positive control that contains each of the biomarkers that is to be tested, or may comprise multiple positive controls that each contain a biomarker or a subset of the group of biomarkers that is to be tested by the method described herein.

In certain embodiments, the kit of parts comprises information about distinct IGFBP2 expression levels that may be detected by the method described herein and guidance on their interpretation. In certain embodiments, the kit of parts comprises one or more reagents for performing an enzymatic activity assay. In certain embodiments, the kit of parts may comprise means to extract and/or isolate RNA. Optionally, the kit of parts comprises at least one primer pair for performing a polymerase chain reaction. Optionally, the kit of parts comprises at least a polymerase, preferably a DNA polymerase in an amount sufficient for conducting a polymerase chain reaction.

The physical form or physical representation of the internal standard in the kit of parts is not particularly limiting for the invention. Therefore, the internal standard may be comprised in the kit of parts as liquid, powder, or a combination of a liquid and a powder. Optionally, the internal standard may be lyophilized. In such embodiments, the kit of parts may comprise a suitable liquid or solution that allows reconstitution prior to usage of the internal standard.

The in vitro methods or kits disclosed herein for detecting IGFBP2 expression are additionally of particular interest for evaluating the effect of a hypothesized pharmaceutically active ingredient, or the effect of each one of a large amount of pharmaceutically active ingredients on thoracic aortic aneurysm development in screening studies.

While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing description. Accordingly, it is intended to embrace all such alternatives, modifications, and variations as follows in the spirit and broad scope of the appended claims. The herein disclosed aspects and embodiments of the invention are further supported by the following non-limiting examples.

EXAMPLES

Example 1. Transcriptomic analysis of aortic tissue samples of TAA mouse models reveals upregulated Igfbp2 as a pan-TAA marker

Materials and methods

RNA isolation and quality control

16-week-old Sv 129 & C57B16N Ipo8t^l~ as well as C57B16J F ^C1O41G/+ mice were euthanized using CCL-inhalation. Ascending aortic tissue was collected, of which RNA was isolated with the RNeasy mini kit (Qiagen). Subsequent cDNA synthesis was done using the SuperScript III First-Strand Synthesis kit. RNA concentrations were determined with the Qubit RNA BR Assay Kit, whereas RNA integrity numbers (RINs; >9.5) were defined with the Fragment Analyzer (Advanced analytics). mRNA sequencing mRNA-sequencing of aortic RNA of TAA-presenting C57B16N Ipo8~ ~ and C57B16J Fw7^cl041G/+ mice was outsourced to Novogene (Cambridge, UK). Sequence capture involved the NEBNext® Ultra RNA Eibrary Prep Kit and sequencing (9M reads) was done on a NovaSeq 6000 system (Illumina). The paired-end reads were preprocessed with trimmomatic 0.39 and aligned to GRCm38 build 102 ENSEMBL with STAR 2.7.5c. Gene expression quantification was done with featureCounts v2.0. 1 and for differential gene expression analysis of the protein coding genes (patients versus controls), DESeq2 1.26 was used. Gene set enrichment analysis (GSEA) was performed with the R package fGSEA 1.12.

RT-qPCR

Alterations in aortic gene expression levels of Igfbp2 were studied using SYBR green (SYBR Green no ROX mix, Eurogentec) chemistry on a Bio-Rad CFX_384 system. Two reference genes (Rpl4 and Rpl32) were used for normalization. Primers were designed with Primer3, after which amplification efficiency and primer specificity were verified based on the determination coefficient R2, Ct range and melting curve. An amplification efficiency of 85% and 110% was reached for all primer sets. Primer sequences are available upon request.

Results

Bulk mRNA-sequencing of aortic tissue samples of 16-week-old TAA-presenting male C57B16J F W7^C1041G/+ and C57B16N Ipo8~'~ mice as well as their respective wild-type littermates revealed a significant upregulation of Igfbp2 in both transgenic models, which we confirmed with RT-qPCR (Figure 1). Exploration of mRNA-sequencing data of aortic samples of other TAA-presenting mice (in collaboration with Erasmus MC, Rotterdam) as well as a literature search on RNA-profiling experiments in transgenic TAA mice exposed increased Igfbp2 mRNA expression in four more TAA models (i.e. Smad3~^l Fbnl^msBJmsR, Efemp2'¹' andZox^{- -}); three syndromic and one non-syndromic mouse models, respectively. Importantly, RT-qPCR revealed normal Igfbp2 expression in the aorta of 16- week-old male 129Sv7poS ^/_ mice (Figure 1). These mice carry the exact same gene defect as the TAA- presenting C57B16N IpoF'- mice, but do not present with TAA until the age of 52 weeks because of the “protective” Svl29 genetic background. IGFBP2 is an interesting candidate biomarker from a functional point of view too. According to the GTEx bulk tissue gene expression dataset (https://www.gtexportal.org), it is highest expressed in the aorta. Moreover, investigations in animal and cell models have revealed a role for Igfbp2 in V SMC proliferation/migration and angiogenesis as well as a functional relationship with several molecules that have been linked to TAA development before, including Sirtl, Adamtsl and pErkl/2. Along with the fact that IGFBP2 is secreted into the blood stream, our murine data provides interesting preliminary data suggesting that Igfbp2 is a novel well-accessible TAA biomarker.

Example 2. Serum IGFBP2 protein levels differ between TAA patients, acute aortic dissection patients and control individuals.

Materials and methods

Patient and control sample collection

Samples from TAA patients (N=104; via genetic, cardiology or cardiothoracic outpatient clinic visits), acute aortic dissection cases (N=3; via cardiothoracic surgery hospitalized patients) and control individuals (N=20; via cardiology department (after normal echocardiography) have been collected at the University hospital of Antwerp. Sample collection is still ongoing with average recruitment rate of 3 patients per week. Venous blood samples were drawn from an antecubital vein and collected in EDTA, serum separator and PAX tubes. EDTA samples were processed within 30 minutes after collection for preservation of plasma. Serum, plasma and PAX tubers are stored at -80°C for batch analysis.

ELISA IGFBP2 levels in serum samples of 20 TAA, 3 acute aortic dissection and 14 control individuals were measured using the R&D systems quantikine IGFBP2 ELISA kit.

Results

The first IGFBP2 ELISA test pointed out that, in a statistical model where correction for age is applied, a ROC curve with an AUC of 0,876 could be obtained (Figure 2). Additionally, remarkably high levels of IGFBP2 were observed in the serum samples of acute aortic dissection patients.

The predictive value of IGFBP2 was found to be stronger for thoracic aortic dissection than for thoracic aortic aneurysm. The results for dissection patients are depicted in Figure 3.

Claims

1. An in vitro method for the detection of thoracic aortic aneurysm (TAA) and/or thoracic aortic dissection (TAD) in a subject, said method comprising determining the level of insulin-like growth factor binding protein 2 (IGFBP2) expression in a biological sample of said subject.

2. The method according to claim 1, wherein the biological sample is a serum sample of the subject.

3. The method according to claim 1 or 2, wherein said method comprises comparing said level of IGFBP2 expression in a biological sample and determining whether said IGFBP2 level is altered when compared to a control sample.

4. The method according to any one of the preceding claims, wherein the method comprises determining whether the IGFBP2 level is increased when compared to a control sample.

5. The method according to any one of the preceding claims, wherein the control sample is a sample obtained from an individual having normal aortic diameters or no aortic dissection.

6. The method according to any one of the preceding claims, wherein said method is a method of detecting asymptomatic TAA, asymptomatic TAD, and/or symptomatic TAD.

7. The method according to any one of the preceding claims, wherein said method is a method of monitoring disease progression in a subject diagnosed with TAA.

8. The method according to any one of the preceding claims, wherein said method is a method of predicting aortic dissection in said subject.

9. The method according to any one of the preceding claims, further comprising determining whether said subject has a genetic predisposition to developing TAA and/or TAD.

10. The method according to any one of the preceding claims, wherein said subject is a subject with bicuspid aortic valves (BAV), idiopathic degenerative disease or a tricuspid aortic valve (TAV).

11. The method according to any one of the preceding claims, further comprising detecting one or more of additional biomarkers in the biological sample of said subjects.

12. The method according to any one of the preceding claims, further comprising treating said subject with medicaments effective against TAA progression and optionally assessing their efficacy by means of IGFBP2 expression level monitoring.

13. The method according to any one of the preceding claims, wherein said method comprises determining the level of Igfbp2 protein and/or the level of IGFBP2 mRNA present in said biological sample.

14. The method according to claim 13, wherein the level of IGFBP2 is detected by a biochemical method, immunoassay method, mass spectrometry analysis method, chromatography method, or combinations thereof.

15. Insulin-like growth factor binding protein 2 (IGFBP2) for use in the detection of thoracic aortic aneurysm development or thoracic aortic dissection development.